Multiple cytokine protein complexes

ABSTRACT

The invention relates to protein complexes and fusion proteins including at least two different cytokine molecules. The protein complexes and fusion proteins may further include a targeting moiety such as a region of an immunoglobulin. Methods of using the protein complexes and fusion proteins are also disclosed.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/634,368, filedAug. 9, 2000, now U.S Pat. No. 6,617,135, which claims the benefit ofU.S. Ser. No. 60/147,924, filed Aug. 9, 1999, the disclosures of each ofwhich are herein incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to methods for the construction andexpression of multiple cytokine protein complexes and theircompositions. More specifically, the invention relates to fusionproteins composed of multiple cytokines and a targeting component, andmethods of using the same for the treatment of diseases such as cancerand viral infection.

BACKGROUND OF THE INVENTION

The regulatory networks controlling the immune system rely on secretedprotein signaling molecules termed cytokines to turn on and off thefunctions of immune cells as well regulate their proliferation. Theseresponses generally involve multiple cytokines that act in concert toachieve the desired biological effect. Certain cytokines such asinterleukin-2 (IL-2) can induce immune cell proliferation by themselvesand can activate other functions including secondary cytokine secretion.Another cytokine, interleukin-12 (IL-12) [reviewed by Trinchieri, 1994,Blood 84:4008–4027], can induce proliferation of certain immune cellsand induce another key immune modulator, interferon-γ (IFN-γ). Thisinduction of IFN-γ is a key activity of IL-12, although IL-12 has otherimportant activities that are IFN-γ independent. Since IL-12 itself isinduced at an early stage in infectious disease situations, it isthought to link the innate and acquired immune systems.

Many in vitro studies with both mouse and human immune cells have shownthe importance of cytokine combinations in the development of optimalimmune responses. For example, most T cells do not express IL-12receptors (IL-12R) until they have been activated with mitogens orcultured in high concentrations of IL-2 [Desai et al. (1992), J.Immunol. 148:3125–3132]. Once the receptors are expressed, the cellsbecome far more responsive to IL-12. Furthermore, IL-12 induces IFN-γtranscription, but IFN-γ mRNA is degraded shortly thereafter, in thepresence of IL-2, the mRNA is stabilized, resulting in a dramaticincrease in the amount of IFN-γ produced [Chan et al. (1992) J. Immunol.0.148:92–98]. In other studies, it was found that the cytokinecombinations IL-3 plus IL-11 or IL-3 pus Steel Factor had a synergisticeffect with IL-12 on the proliferation of early hematopoietic progenitorcells [Trinchieri, 1994; cited above]. The combination of interleukin-4and GM-CSF is particularly useful in stimulating dendritic cells(Palucka et al. [1998] J. Immunology 160:4587–4595). For stimulation ofthe cell-mediated immune response, it is also useful to combine IL-12with IL-18, a recently discovered Th1-promoting cytokine with someactivities that are complementary to IL-12 (Hashimoto et al. [1999] J.Immunology 163:583–589; Barbulescu et al. [1998] J. Immunology160:3642–3647). In addition, IL-2 and interferon-γ are synergistic incertain circumstances [Palladino, M. A., U.S. Pat. No. 5,082,658].

In many of these synergy studies it was found that the relative level ofeach cytokine was very important. Whereas the addition of IL-12 in thepresence of suboptimal amounts of IL-2 led to synergy in the inductionof proliferation, cytolytic activity and IFN-γ induction, combinationsof IL-2 and IL-12 using a high dose of one cytokine were found to beantagonistic [Perussia et al., J. Immunol. 149:3495–3502 (1992);Mehrotra et al., J. Immunol. 151:2444–2452 (1993)]. A similar situationalso exists in combinations of IL-12 and IL-7.

Synergy studies between IL-12 and other cytokines for the generation ofanti-tumor responses in mice have also shown mixed results. In somemodels synergy was seen at suboptimal doses of each cytokine and higherdoses led to enhanced toxicity, while in other models, combinations ofIL-12 and IL-2 showed little or no synergy [see, for example, Nastala etal., J. Immunol. 153:1697–1706. (1994)]. These results may reflect theinherent difficulty of combining two potentially synergistic agents invivo, especially when there is the need to maintain a fixed ratio ofactivities of two agents with different pharmacological properties, suchas different circulating half-life and biodistribution.

In in vitro cell culture experiments, it is straightforward to controlcytokine levels, but many factors can affect the relativebiodistribution and localization of cytokines in vivo, thus affectingtheir immunostimulatory capacity. The most important of these factors isthe half-life. The half-life of IL-2 in the circulation after bolusinjection is approximately 10 minutes. In striking contrast to thesepharmacokinetic properties, the circulating half-life of IL-12 has beenreported to be >3 hr in mice [Wysocka et al (1995) Eur. J. Immunol.25:672] and from 5–10 hr in humans [Lotze et al. (1996) Ann NY Acad Sci795:440–454].

This difference is thought to be due to the relatively small sizes ofboth IL-2 and GM-CSF (15–25 kD vs. 75 kD for IL-12), allowing IL-2 andGM-CSF to be cleared by renal filtration. Proteins with a molecularweight of less than about 50 kD are cleared by renal filtration. Almostall cytokines are smaller than 50 kD and undergo similar, rapidclearance by renal filtration. When treatment with two such small,rapidly cleared cytokines is desired, it is sufficient to simplyco-administer the cytokines. However, co-administration is not optimalfor cytokines with significantly different half-lives.

The systemic administration of cytokines is difficult due to theirdeleterious side effects. For example, high levels of Interferon-alpharesult in significant side effects, including skin, neurologic, immuneand endocrine toxicities. It is expected that multiple cytokine fusionsmight show particularly serious side effects.

To reduce side effects of systemic administration of cytokines, onestrategy is to fuse a cytokine to a second molecule with targetingcapability. Fusions in which an Fc region is placed at the N-terminus ofa another protein (termed ‘immunofusins’ or ‘Fc-X’ fusions, where X is aligand such as Interferon-alpha) have a number of distinctive,advantageous biological properties [Lo et al., U.S. Pat. Nos. 5,726,044and 5,541,087; Lo et al., Protein Engineering 11:495]. In particular,such fusion proteins can still bind to the relevant Fc receptors on cellsurfaces. However, when the ligand binds to its receptor on a cellsurface, the orientation of the Fc region is altered and the sequencesthat mediate antibody-dependent cell-mediated cytotoxicity (ADCC) andcomplement fixation appear to be occluded. As a result, the Fc region inan Fc-X molecule does not mediate ADCC or complement fixationeffectively. The cytotoxic effect due to the fusion of an N-terminalcytokine and a C-terminal Fc region is well known. For example, fusionof IL-2 to the N-terminus of an Fc region creates a molecule that isable to bind to cells bearing the IL-2 receptor, fix complement, andlyse the cells as a result [Landolfi, N. F. (1993) U.S. Pat. No.5,349,053]. In contrast, Fc-IL-2 fusion proteins do not have thisproperty. Thus, Fc-X fusions are expected to have the virtues ofincreased serum half-life and relative concentration in the liver,without the deleterious effects of ADCC and complement fixation.

It has been demonstrated that many different proteins with short serumhalf-lives can be fused to an Fc region in an Fc-X configuration, andthe resulting fusions have much longer serum half-lives. However, theserum half-lives of two different Fc fusions will not generally beidentical. Thus, when delivery of two different X moieties is desired,co-administration of two different Fc-X proteins will not generally beoptimal.

Under some circumstances, a better approach is to target the effect ofthe cytokine to a cell surface antigen by fusing it to an antibody (orfragment derived therefrom) having specificity and affinity for thatantigen (Gillies, U.S. Pat. No. 5,650,150; Gillies et al., Proc. Natl.Acad. Sci. 89:1428) or by linking a protein antigen and stimulatorycytokine via a peptide linkage in the form of a fusion protein (Hazamaet al, Vaccine 11:629). While antibodies themselves can increase thehalf-life of a fused cytokine, there are still differences betweendifferent cytokine fusions with the same antibody [see, for example,Gillies et al., Bioconjugate Chem. 4:230–235 (1993); Gillies et al., J.Immunol. 160:6195–6203] that would make co-localization at a target sitedifficult. As discussed above, this could lead to an imbalance incytokine activities and decrease the desired synergistic effects. Inaddition, the use of two different fusion proteins requires testing eachfusion separately for its safety and effectiveness profile, and thenfurther testing as mixtures.

SUMMARY OF THE INVENTION

The present invention provides complexes or fusions between two or moredifferent cytokines, which are useful for general as well as targetedimmune therapy. These complexes or fusions optionally include otherprotein moieties. One feature of such complexes or fusions is that theyprovide the activities of the component cytokines in a fixed ratio.

Generally, the invention relates to a protein complex containing atleast two different cytokines. The cytokines could be in the samepolypeptide chain or connected by a covalent bond such as a disulfidebond or a bond formed by chemical crosslinking. Alternatively, thecytokines could be in a stable, non-covalent association. In somepreferred embodiments, the protein complex comprises a targeting moietysuch as an antibody or antibody fragment, that targets the complex to alocus in a mammal.

In a preferred embodiment, the invention provides a protein complexcombining the bioactivity of a two-chain cytokine, such as IL-12, withthat of a second cytokine. The cytokines may be covalently bonded (e.g.fused) to each other. The cytokines may also be associated through othermoieties. For example, the polypeptide chain containing the secondcytokine could include a binding moiety that specifically binds IL-12,such as an antibody to IL-12 or a receptor to IL-12. Alternatively, thebinding moiety could interact with a second moiety that is associatedwith the IL-12. For example, if a polypeptide chain encoding a subunitof IL-12 also includes avidin, the polypeptide containing the secondcytokine may include biotin as a targeting moiety. In one preferredembodiment, the second cytokine is IL-2.

The invention provides methods for the production of fusion proteins ofIL-12 that maintain both IL-12 activity and that of the second cytokine,while providing a longer, single pharmacokinetic behavior, similar tothat of IL-12 itself, that increases the duration of the activity of thesecond cytokine and maintains the balance of activities of the twocytokines after injection into an animal.

In another embodiment of the invention, the fusion proteins comprise aheterodimeric form of IL-12 in which the p35 and p40 subunits of IL-12are linked by a disulfide bond and covalently bonded to a secondcytokine at either the amino or carboxyl terminus of the p35 or p40subunit of IL-12 with the general formula IL-12-X or X-IL-12, where X isa second cytokine.

In another embodiment of the invention, the fusion proteins comprise asecond cytokine covalently bonded at either the amino or carboxylterminus to a single-chain (sc) form of IL-12 comprising the twopolypeptide subunits joined via a flexible peptide linker with thegeneral formula scIL-12-X or X-scIL-12.

In yet another embodiment, two cytokines are further fused to a proteincapable of forming a dimeric or multimeric structure, at either theamino or carboxyl terminus of said protein chain. In a preferred form ofthis embodiment, one of the fusion protein forms of IL-12 with a secondcytokine is further fused to a portion of an immunoglobulin (Ig) chain,such as the Fc region, that is capable of dimerization. Furtherembodiments include fusion of at least one polypeptide chain of IL-12 ateither terminus of a portion of an Ig chain and a second cytokine fusedat the other terminus.

In another embodiment, two or more cytokines are fused to a protein withtargeting capability by virtue of binding to a specific receptor. Forexample, an Fc region is capable of binding to Fc receptors, which areabundant in the liver. Fusions of an Fc region with multiple cytokinesillustrate the advantages of both dimerization and targeting, but insome circumstances it is useful to construct fusions of multiplecytokines that have only multimerization or targeting capability, butnot both capabilities.

In yet another embodiment, a fusion protein comprising multiplecytokines is further fused at either the amino or carboxyl terminus to amember of a class of molecules with diverse targeting capability, suchas an antibody or a peptide aptamer with or without a scaffold (Colas etal. [1998] Proc Natl Acad Sci USA. 95:14272–7). A particular embodimentis the fusion of multiple cytokines to at least a portion of an antibodycapable of binding an antigen, such as an intact antibody, asingle-chain antibody, or a single-chain Fv region. Further embodimentsinclude fusions of at least one polypeptide chain of IL-12 at eitherterminus of at least a portion of an antibody chain that is capable ofbinding an antigen, and a second cytokine fused at the other terminus.

According to the above descriptions, it is generally preferred toconstruct multiple cytokine fusion proteins and multiplecytokine-antibody fusion proteins by genetic engineering techniques,such that the component proteins are linked by covalent bonds such asamide bonds or disulfide bonds. However, it is also useful to usechemical cross-linkers to construct such protein complexes. Such methodsare well established in the art of protein chemistry. Alternatively, itis sometimes sufficient to generate protein complexes by fusingdifferent cytokines with partner proteins that form stable non-covalentcomplexes. For example, a non-covalent heterodimer support protein isused: a first cytokine is fused to one subunit of the heterodimer, asecond cytokine is fused to a second subunit of the heterodimer, and thetwo fusion proteins are mixed under appropriate conditions. For example,nucleic acids encoding the two subunit-cytokine fusion proteins areexpressed in the same cell. In this way, a multiple cytokine proteincomplex may be constructed in which the component cytokines are notcovalently linked, directly or indirectly. To achieve the purpose of theinvention, it is necessary that such a complex is stable enough to bemaintained upon administration of an animal and achieve a biologicaleffect.

The invention also provides nucleic acids that encode fusion proteinscomprising two or more cytokines, where one of the cytokines ispreferably IL-12 and the fusion protein encoded by the nucleic acidoptionally includes other protein moieties. Preferred embodimentsinclude nucleic acids that encode fusions of two or more cytokines to adimerizing protein, such as an Fc portion of an antibody chain. Anotherset of preferred embodiments are nucleic acids that encode fusions oftwo or more cytokines to a protein with targeting capability, such as anantibody.

The invention also provides methods for construction of fusions of twoor more cytokines, as well as methods for expression of such fusionproteins.

The invention also provides methods for treatment of diseases and othermedical conditions, in which treatment involves the useful combinationof the activity of two or more proteins. In one embodiment, at least oneof the proteins has a short (e.g. less than 20 minutes) or onlymoderately long (e.g. less than 40 minutes) serum half-life. Theproteins are fused by genetic engineering or other techniques andadministered to a human or animal. In this way, the activities of thetwo proteins are present in a fixed ratio, and separate administrationson different dosing schedules of the two proteins are not required. Inaddition, the serum half-life of the fusion protein will generally bemore similar to that of the protein component with the longer serumhalf-life, thus lengthening the effective half-life of the protein orproteins with the shorter serum half-life.

More specifically, the invention provides methods of immune-therapeutictreatment of diseases, such as cancer or infections or other diseases,that might be usefully treated with a two-chain cytokine such as IL-12in combination with a second cytokine. In a preferred embodiment, IL-12is fused with IL-2 or GM-CSF and administered to an animal or human. Inother preferred embodiments, GM-CSF is fused to IL-4 and administered toan animal or a human. In another embodiment, IL-12 is fused to IL-18 andadministered to an animal or a human. Such treatments can be used incombination with other disease treatments. In addition, the inventionprovides methods of vaccination against diverse antigens, which can beused to prevent or treat various diseases.

In other embodiments of these methods, two different cytokines are fusedto a dimeric protein moiety, such as the Fc region of an antibody, andare administered to an animal or human. In a preferred form of thesemethods, the cytokine IL-12 is fused to the Fc region along with asecond cytokine that is more preferably IL-2 or GM-CSF.

In yet other embodiments of these methods, two different cytokines arefused to an intact antibody, and are administered to an animal or human.In a preferred form of these methods, the cytokine IL-12 is fused to theantibody moiety along with a second cytokine that is more preferablyIL-2 or GM-CSF. The invention also discloses mixtures ofantibody-cytokine fusion proteins that are useful in treating diseases.In one embodiment, a mixture of an antibody-IL-2 fusion protein and anantibody-IL-12 fusion protein is used to treat disease. For example,cancer, viral infection, or bacterial infection is treated.

BRIEF DESCRIPTION OF THE DRAWINGS

The preceding and other objects of the present invention, and thevarious features thereof, may be more fully understood from thefollowing descriptions, when read together with the accompanyingdrawings. Throughout the drawings, like numbers refer to likestructures.

FIG. 1A schematically illustrates the fusion of two cytokines in itssimplest form: one cytokine is fused to a second cytokine, optionallythrough a linker. FIGS. 1B–1I show various ways in which a secondcytokine (labeled ‘cyt’) may be attached to the heterodimeric cytokineIL-12. Specifically, the second cytokine can be fused to the C-terminusof p40 (FIG. 1B), the N-terminus of p40 (FIG. 1C), the C-terminus of p35(FIG. 1D) or the N-terminus of p35 (FIG. 1E). In addition, FIG. 1 showshow a second cytokine may be fused to a single chain version of IL-12.Specifically, single chain IL-12 molecules may have p35 N-terminal top40, with the second cytokine at the C-terminus (FIG. 1F) or theN-terminus (FIG. 1G). Alternatively, single chain IL-12 molecules mayhave p40 N-terminal to p35, with the second cytokine at the C-terminus(FIG. 1H) or the N-terminus (FIG. 1I).

FIGS. 2A–2C schematically show how multiple-cytokine fusions (boxed)depicted in FIG. 1 may be further fused to an Fc region of an antibody,shown here as a hinge (H), a CH2 domain and a CH3 domain (ovals).Specifically, any of the eight molecules of FIG. 1 may be fused toeither the C-terminus (FIG. 2A) or N-terminus (FIG. 2B) of the Fcregion. In addition, the first cytokine and second cytokine (each boxed)need not be directly attached to each other, but can be connectedthrough the Fc moiety (FIG. 2C).

FIGS. 3A–3G schematically show a subset of the ways in which a multiplecytokine fusion protein may be further fused to an intact immunoglobulinsuch as an IgG. The heavy chain V region is shown as an oval labeledV_(H), the light chain V region is shown as an oval labeled V_(L), andconstant regions are blank ovals. A multiple cytokine fusion, asillustrated in FIG. 1, may be placed at the C-terminus of the heavychain (FIG. 2A), the N-terminus of the heavy chain (FIG. 2B), theN-terminus of the light chain (FIG. 2C), or the C-terminus of the lightchain (FIG. 2D). In addition, there are many ways in which a first andsecond cytokine could be separately attached at the N- and C-termini ofthe heavy and light chains; three of these are shown in FIGS. 3E–3G.

FIGS. 4A–4C schematically show how a first cytokine and a secondcytokine may be fused to a “single-chain” antibody in which the variablelight and variable heavy chains are fused, and the protein is expressedas a single polypeptide that then homodimerizes. Specifically, amultiple cytokine fusion may be placed at the C-terminus (FIG. 4A), orthe N-terminus (FIG. 4B). In addition, the a first cytokine and secondcytokine need not be directly attached, but can be connected through thesingle-chain antibody moiety (FIG. 4C).

FIGS. 5A–5C schematically show how a first cytokine and a secondcytokine may be fused to a single-chain Fv region consisting of thefused variable regions from a heavy chain and a light chain.Specifically, a first cytokine-cytokine fusion may be placed at theC-terminus (FIG. 5A), or the N-terminus (FIG. 5B). In addition, thefirst cytokine and second cytokine need not be directly attached, butcan be connected through the single-chain Fv moiety (FIG. 5C).

FIGS. 6A and 6B show the synergy between IL-12 and IL-2 in the inductionof IFN-γ by human peripheral blood mononuclear cells (PBMCs) in responseto the separate cytokines or fusion proteins. In FIG. 6A, cells weretreated with human IL-12 before (squares) or after phytohemagglutininactivation (X's), or with IL-12-IL-2 fusion protein before (diamonds) orafter phytohemagglutinin activation (triangles). FIG. 6B shows anexperiment in which cells were treated with a mixture of IL-12 plus IL-2added in a 1:1 molar ratio (black diamonds), human Fc-IL-12-IL-2 fusionprotein (gray squares), and human antibody-IL-12-IL-2 fusion protein(light gray triangles). The X axis indicates the concentration of IL-12in μg/ml, whether present as an intact protein or as a fusion protein.The y-axis indicates IFN-γ concentration (in ng/ml), which was assayedby ELISA.

FIG. 7 shows a typical IL-12 bioassay that separately measures activityof a fusion protein and compares it to that of a non-fused IL-12molecule. What is depicted is the stimulation of ³H-thymidine uptake ofhuman PBMCs in response to murine IL-12 (white circles), to a mixture ofmurine IL-12 plus IL-2 added in a 1:1 molar ratio (black squares), tomurine IL-2 (white triangles), and to an antibody-murine IL-12-IL-2fusion protein (black diamonds). The X axis indicates the concentration(pM) of monomeric cytokine(s), whether present as an intact protein oras a fusion protein; the y-axis indicates cpm of tritiated thymidineincorporation.

FIG. 8 shows a standard IL-2 bioactivity assay. The graph shows thestimulation of mouse CTLL cell proliferation, in response to murine IL-2(circles), to an antibody-murine IL-12-IL-2 fusion protein (diamonds),and murine IL-12 (squares). The x axis indicates the concentration (pM)of monomeric cytokine(s), whether present as an intact protein or as afusion protein. Cells were incubated in medium containing variousamounts of cytokine or fusion protein for 48 hours, then assayed forviable cell number using the MTT/MTS assay. The y-axis indicates theabsorbance at 490 nanometers in units of optical density (OD).

FIG. 9 shows the stimulation of ³H-thymidine uptake by human PBMCs inresponse to murine IL-12 (white circles), to a mixture of murine IL-12plus IL-2 added in a 1:1 molar ratio (black circles), to a murineFc-single-chain-IL-12-IL-2 fusion protein (black triangles), and tomurine single-chain IL-12 fused to murine IL-2 (black diamonds). The xaxis indicates the concentration (pM) of monomeric cytokine(s), whetherpresent as an intact protein or as a fusion protein; the y-axisindicates cpm of tritiated thymidine incorporation.

FIG. 10 shows the stimulation of ³H-thymidine uptake by human PBMCs inresponse to murine IL-12 (white circles), to a mixture of murine IL-12plus GM-CSF added in a 1:1 molar ratio (black circles), to murine GM-CSF(black triangles), and to an murine Fc-murine IL-12-GM-CSF fusionprotein (X's). The x axis indicates the concentration (pM) of monomericcytokine(s), whether present as an intact protein or as a fusionprotein; the y-axis indicates cpm of tritiated thymidine incorporation.

FIG. 11 shows the effect of antibody-cytokine-cytokine fusion proteintreatment of Balb/C mice bearing subcutaneous tumors derived from CT26colon carcinoma cells that were engineered to express human EpCAM, theantigen for KS-1/4. Black diamonds indicate average tumor volumes inmice that were injected with PBS as controls on days 0, 1, 2, 3, and 4.Triangles indicate average tumor volumes in mice treated with 6micrograms of KS-IL-12-IL-2. Squares indicate average tumor volumes inmice treated with 3.4 micrograms of KS-IL2 and 5.3 micrograms ofKS-IL12. Intratumoral injections were performed. The x-axis indicatesthe number of days elapsed following the first injection; the y-axisindicates the average tumor volume in cubic milliliters.

FIG. 12 shows the effect of antibody-cytokine-cytokine fusion proteintreatment of SCID mice bearing subcutaneous tumors derived from CT26colon carcinoma cells that were engineered to express human EpCAM.Diamonds indicate average tumor volumes in mice that were injected withPBS as controls on days 0, 1, 2, 3, and 4. Triangles indicate averagetumor volumes in mice treated with 6 micrograms of KS-IL-12-IL-2.Squares indicate average tumor volumes in mice treated with 3.4micrograms of KS-IL2 and 5.3 micrograms of KS-IL12. Intratumoralinjections were performed. The x-axis indicates the number of dayselapsed following the first injection; the y-axis indicates the averagetumor volume in cubic milliliters.

FIG. 13 compares the effect of antibody-cytokine andantibody-cytokine-cytokine fusion protein treatment of mice bearingsubcutaneous tumors of Lewis lung carcinoma (LLC) cells that wereengineered to express human EpCAM. Diamonds indicate average tumorvolumes in mice that were injected intratumorally with PBS as controlson days 0, 1, 2, 3, and 4. Squares indicate average tumor volumes inmice injected intratumorally with 20 micrograms of KS-IL2 on days 0, 1,2, 3, and 4. Triangles indicate average tumor volumes in mice injectedintratumorally with 20 micrograms of KS-IL12 on days 0, 1, 2, 3, and 4.X's indicate average tumor volumes in mice injected intratumorally with20 micrograms of KS-IL-12-IL-2 on days 0, 1, 2, 3, and 4. The x-axisindicates the number of days elapsed following the first injection; they-axis indicates the average tumor volume in cubic milliliters.

FIG. 14 shows the effect of antibody-cytokine-cytokine fusion proteintreatment of mice bearing subcutaneous tumors derived from Lewis lungcarcinoma cells that were engineered to express human EpCAM. Diamondsindicate average tumor volumes in mice that were injected with PBS ascontrols on days 0, 1, 2, 3, and 4. Triangles indicate average tumorvolumes in mice treated with 20 micrograms of KS-IL-12-IL-2. Squaresindicate average tumor volumes in mice treated with 11.5 micrograms ofKS-IL2 and 18 micrograms of KS-IL12. Intratumoral injections wereperformed. The x-axis indicates the number of days elapsed following thefirst injection; the y-axis indicates the average tumor volume in cubicmilliliters.

FIG. 15 shows the effect of antibody-cytokine-cytokine fusion proteintreatment of mice bearing subcutaneous tumors derived from Lewis lungcarcinoma cells that either do or do not express human EpCAM. Blacksquares indicate average tumor volumes in mice bearing LLC/KSA-derivedtumors. Black diamonds indicate average tumor volumes in mice bearingLLC-derived tumors. Mice were treated with 20 micrograms of KS-IL12-IL2on days 0, 1, 2, 3, and 4. Intratumoral injections were performed. Thex-axis indicates the number of days elapsed following the firstinjection; the y-axis indicates the average tumor volume in cubicmilliliters.

FIG. 16 shows the effect of antibody-cytokine-cytokine fusion proteintreatment of mice bearing subcutaneous tumors derived from Lewis lungcarcinoma cells. About 10⁶ cells were injected subcutaneously on Day 0.Diamonds indicate average tumor volumes in naive mice. Squares indicateaverage tumor volumes in mice that had previously had subcutaneoustumors derived from Lewis lung carcinoma cells that were engineered toexpress human EpCAM, and had been cured of these tumors by treatmentwith KS-IL12-IL2. The x-axis indicates the number of days elapsedfollowing the injection; the y-axis indicates the average tumor volumein cubic milliliters.

FIGS. 17A and 17B show the effect of single- or multiple-cytokineprotein secretion by tumor cells on the ability of the cells to formtumors in an animal with a normal immune system. In FIG. 17A, four setsof mice are compared: C57BL/6 mice injected s. c. with 1×10⁶ LLC tumorcells (black diamonds); C57BL/6 mice injected s. c. with 5×10⁶ LLC tumorcells (white diamonds); C57BL/6 mice injected s. c. with 1×10⁶ LLC tumorcells expressing scIL-12 (black triangles); and C57BL/6 mice injected s.c. with 5×10⁶ LLC tumor cells expressing scIL-12 (white triangles). FIG.17B compares C57BL/6 mice injected s. c. with 1×10⁶ LLC tumor cells(black diamonds); C57BL/6 mice injected s. c. with 5×10⁶ LLC tumor cells(white diamonds); C57BL/6 mice injected s. c. with 1×10⁶ LLC tumor cellsexpressing scIL-12-IL-2 (X's); and C57BL/6 mice injected s. c. with5×10⁶ LLC tumor cells expressing scIL-12 (white circles). The x-axisindicates number of days after injection of the tumor cells. The y-axisindicates the tumor volume in cubic millimeters.

FIG. 18 shows the effect of single- or multiple-cytokine proteinsecretion by tumor cells on the ability of the cells to form tumors inan immune-deficient animal. This Figure compares SCID mice injected s.c. with 1×10⁶ LLC tumor cells (black diamonds); SCID mice injected s. c.with 1×10⁶ LLC tumor cells expressing scIL-12 (black triangles); andSCID mice injected s. c. with 1×10⁶ LLC tumor cells expressing scIL-12(white circles). The x-axis indicates the number of days after injectionof the tumor cells. The y-axis indicates the tumor volume in cubicmillimeters.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides protein molecules in which two or more distinctcytokines are fused or complexed. The protein complexes or fusionproteins may optionally include additional protein moieties, includingmoieties capable of multimerization and targeting such as antibody Fcregions and antibody regions that include antigen combining sites. Theinvention also provides nucleic acids encoding multiple-cytokine fusionproteins. The invention also provides methods for the construction ofnucleic acids encoding multiple-cytokine fusion proteins, methods forproduction of multiple-cytokine fusion proteins, and methods for use ofmultiple-cytokine fusion proteins in treatment of diseases and medicalconditions.

As used herein, “cytokine” refers to a secreted protein or activefragment or mutant thereof that modulates the activity of cells of theimmune system. Examples of cytokines include the interleukins,interferons, chemokines, tumor necrosis factors, colony-stimulatingfactors for immune cell precursors, and so on.

As used herein, “heterodimeric cytokine” refers to a cytokine consistingof two distinct protein subunits. At present, IL-12 is the onlynaturally occuring heterodimeric cytokine that is known. However,artificial heterodimeric cytokines can be constructed. For example, IL-6and a soluble fragment of IL-6R can be combined to form a heterodimericcytokine, as can CNTF and CNTF-R alpha [Trinchieri (1994) Blood84:4008].

As used herein, “interleukin-12” (IL-12) refers to the two-subunitcytokine consisting of a p35 and p40 subunit, or an active single-chainfusion of p35 and p40, or a species variant, fragment, or derivativethereof.

As used herein, “interleukin-2” (IL-2) refers to any mammalian IL-2,such as human IL-2, mouse IL-2, or an active species or allelic variant,fragment or derivative thereof.

As used herein, “GM-CSF” refers to a mammalianGranulocyte/Monocyte-Colony Stimulating Factor cytokine protein, such ashuman GM-CSF, mouse GM-CSF, or an active species or allelic variant,fragment or derivative thereof.

As used herein, “immunoglobulin Fc region” means the carboxyl-terminalportion of an immunoglobulin heavy chain constant region, or an analogor portion thereof. For example, an immunoglobulin Fc region of IgG maycomprise at least a portion of a hinge region, a CH2 domain, and a CH3domain. In a preferred embodiment the Fe region includes at least aportion of a hinge region and a CH3 domain. In another preferredembodiment, the Fc region includes at least a CH2 domain and morepreferably also includes at least a portion of a hinge region.

As used herein, “peptide linker” means one or more peptides used tocouple two proteins together (e.g. a protein and an Fc region). Thepeptide linker often is a series of amino acids such as. e.g.,predominantly glycine and/or serine. Preferably, the peptide linker is amixed series of predominantly glycine and serine residues and is about10–15 amino acids in length.

As used herein, the term “multimeric” refers to the stable associationof two or more protein subunits through covalent or non-covalentinteraction, e.g. disulphide bonding.

As used herein, the term “dimeric” refers to a specific multimericmolecule where two protein subunits are associated stably throughcovalent or non-covalent interactions. A stable complex is a complexwith a dissociation rate, or off-rate, of at least several minutes (suchthat the complex would be stable long enough during in vivo use to reacha target tissue and have a biological effect. The Fc fragment itselftypically forms a dimer of the heavy chain fragments comprising aportion of the hinge region, CH2 domain and/or CH3 domain. However, manyprotein ligands are known to bind to their receptors as a dimer. If acytokine X dimerizes naturally, the X moiety in an Fc-X molecule willdimerize to a much greater extent, since the dimerization process isconcentration dependent. The physical proximity of the two X moietiesconnected by Fc would make the dimerization an intramolecular process,greatly shifting the equilibrium in favor of the dimer and enhancing itsbinding to the receptor.

As used herein, “vector” means any nucleic acid comprising a nucleotidesequence competent to be incorporated into a host cell and to berecombined with and integrated into the host cell genome, or toreplicate autonomously as an episome. Such vectors include linearnucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectorsand the like. Non-limiting examples of a viral vector include aretrovirus, an adenovirus and an adeno-associated virus.

As used herein, “gene expression” or “expression of a protein” isunderstood to mean the transcription of the DNA sequence, translation ofthe mRNA transcript, and either secretion of a protein product orproduction of the protein product in an isolatable form.

As used herein, an “immunocytokine” is a fusion protein comprising anantibody and a cytokine, as disclosed in U.S. Pat. No. 5,650,150.

As used-herein, a “leader sequence” is a protein sequence that isattached, usually at the N-terminus, to a second protein sequence, andthat directs the second protein sequence to be secreted from a cell. Theleader sequence is usually cleaved and removed from the second proteinsequence, which becomes the mature protein. The term “leader sequence”is generally synonymous with “signal sequence”.

As used herein, “EpCAM” refers to epithelial cell adhesion molecule(Cirulli et al. [1998] 140:1519–1534), and is synonymous with “KSA,”meaning the antigen bound by the monoclonal antibody KS-1/4. EpCAM is acell surface protein that is abundantly expressed on cancer cellsderived from epithelial cells.

As used herein, “KS-1/4” refers to a particular monoclonal antibody thatbinds to EpCAM.

As used herein, “KS-IL2,” “KS-IL12,” and “KS-IL12-IL2” (and the like)refer to antibody-cytokine fusion proteins consisting of KS-1/4 withinterleukin-2, KS-1/4 with interleukin-12, and KS-1/4 with bothinterleukin-12 and interleukin-2, respectively. Analogously named fusionprotein constructs are also used herein. Because it is possible to fusecytokines at several positions on an antibody molecule, a descriptionsuch as “KS-IL12-IL2” refers to the class of proteins comprising KS-1/4with both interleukin-12 and interleukin-2 fused at any possibleposition, unless explicitly stated otherwise.

As used herein, “14.18” refers to a particular monoclonal antibody thatbinds to the tumor-specific antigen GD2.

Several illustrative embodiments of protein constructs embodying theinvention are illustrated in FIGS. 1–5. Parts of the moleculesdiagrammed in FIGS. 2–5 are labeled 1A–1I, referring to the fusionproteins shown in FIGS. 1A–1I and illustrating that any of the fusionproteins from FIG. 1 can be further fused to other proteins asindicated. Cytokines are shown as rectangles, constant regions ofantibodies are shown as ovals, and the heavy chain variable region andlight chain variable region are shown as labeled ovals.

The present invention describes protein complexes containing twodifferent cytokines and optionally including additional proteinmoieties. A homodimeric cytokine (e.g. interferon alpha, interferonbeta, interferon gamma, IL-5, IL-8, or the like), although it containsmultiple subunits, is nevertheless a single cytokine. Similarly, aheterodimeric cytokine such as IL-12, although it contains subunits thatare different, is a single cytokine. Furthermore, a heterodimeric formof normally homodimeric cytokines, such as a MCP-1/MCP-2 heterodimer, orof two alleles of a normally homodimeric cytokine (e.g., Zhang, J. Biol.Chem. [1994] 269:15918–24) is a single cytokine. The complexes of thepresent invention contain two different cytokines, each of which (e.g.IL-2 and IL-12; IL-4; and GM-CSF; MCP-1 and eotaxin; etc.) is capable ofmodulating an activity of a cell of the immune system.

FIG. 1A depicts a preferred embodiment of the invention: in fusionprotein 10, the C-terminus of a first cytokine 12 is fused to theN-terminus of a second cytokine 14, optionally through a linker region(not shown). In some embodiments of the invention, the protein complexof the invention contains at least two cytokines with significantlydifferent serum half-lives. For example, using a small and a largeprotein will often result in a fusion protein with a circulatinghalf-life characteristic of the larger protein. Therefore, in situationswhere the combined effects of IL-12 and a second cytokine are desired,it would be advantageous to express the two cytokines as a fusionprotein of the general formula: IL-12-X or X-IL-12, where X is a secondcytokine. Two particular advantages are seen. First, the serum half-lifeof the more rapidly cleared cytokine is extended. Second, the serumhalf-lives of both cytokines become very similar to each other.

A two-chain cytokine such as IL-12 can be fused to another cytokine atthe N- or C-terminus of either the chain of the two-chain cytokine. Inone embodiment, a second cytokine is fused at either the N-terminus orC-terminus of either the p35 or p40 subunit of IL-12 (FIGS. 1B–1E). Infusion protein 16 of FIG. 1B, the N-terminus of a first cytokine 12 isfused to the C-terminus of the IL-12 subunit p40 18. Subunit p40 18 isconnected to IL-12 subunit p35 20 by a covalent bond 22. In fusionprotein 24 of FIG. 1C, the N-terminus of p40 subunit 18 is fused to theC-terminus of first cytokine 12 and is connected to p35 subunit 20 by acovalent bond 22. FIG. 1D depicts fusion protein 26 in which theN-terminus of first cytokine 12 is fused to the C-terminus of p35subunit 20, which is connected by covalent bond 22 to p40 subunit 18. InFIG. 1E, fusion protein 28 includes p35 subunit 20, fused at itsN-terminus to the C-terminus of first cytokine 12 and connected bycovalent bond 22 to p40 subunit 18.

In a second embodiment, the subunits of IL-12 may be fused to form asingle-chain protein, scIL-12, with either the p35 subunit or the p40subunit at the N-terminal position; the second cytokine may be attachedto the N- or C-terminus of the resulting scIL-12 (FIGS. 1F–1I). Thus, ina preferred embodiment depicted in FIG. 1F, fusion protein 30 containssingle-chain IL-12 in which the N-terminus of p40 subunit 18 is fused tothe C-terminus of p35 subunit 20, optionally through a peptide linker.In this embodiment, the N-terminus of cytokine 12 is fused to theC-terminus of p40 subunit 18. In the embodiment shown in FIG. 1G, theN-terminus of p35 subunit 20 is fused to the C-terminus of cytokine 12,optionally through a peptide linker. FIGS. 1H and 1I show fusionproteins 34 and 36 containing another single-chain version of IL-12 inwhich the N-terminus of the p35 subunit is fused to the C-terminus ofthe p40 subunit, optionally through a peptide linker. In fusion protein34, shown in FIG. 1H, the N-terminus of cytokine 12 is fused to theC-terminus of p35 subunit 20. In fusion protein 36, shown in FIG. 11,the N-terminus of p40 subunit 18 is fused to the C-terminus of cytokine12. In a highly preferred embodiment, IL-12 is fused to IL-2.

The production of such molecules is further illustrated in the examples.

It is often convenient to express heteromultimeric molecules, such asIL-12 or an antibody, as single-chain molecules in which thenon-identical subunits are connected by short amino acid linkers [Hustonet al (1988) Proc. Nat. Acad. Sci. 85: 5879; Lieschke et al. (1997) NatBiotechnol. 15:35; Lieschke; G. J. and Mulligan; R. C., U.S. Pat. No.5,891,680]. A gene fusion is constructed, and then the desired proteincan be expressed in cells containing a single recombinant DNA construct.Such single-chain versions of a heteromultimeric cytokine can be furtherfused to a second cytokine, which still allows a fusion protein with thedesired activities to be expressed from a single recombinant DNAconstruct. The expression of such molecules is illustrated in theexamples.

The invention also describes a fusion protein comprising IL-4 andGM-CSF. This combination is particularly useful in functionallystimulating antigen presentation by dendritic cells. Another usefulfusion comprises IL-12 and IL-18. These cytokines both promote the Th1response, but have somewhat different, complementary activities.

The invention also describes fusion proteins in which multiple distinct,fused cytokines are further fused to a protein capable of formingmultimers, such as homodimers or heterodimers. The advantage of such amolecule is that the potency of one or more of the cytokines may beenhanced by dimerization. In some cases, enhancement of potency bydimerization can occur because the cytokine binds to its receptor as adimer. In one embodiment, multiple cytokines are fused to a portion ofan antibody molecule, such as an Fc region (FIG. 2). In anotherembodiment, IL-12 and a second cytokine are fused to the homodimerizingprotein moiety. In a preferred embodiment, the second cytokine is IL-2or GM-CSF. The fusion proteins may be created in a variety of ways,reflecting all the various orderings of several distinct proteinmoieties from the N- to C-termini in a fusion protein. For example wheninterleukin-12 and a second cytokine are fused to an Fc region, the twocytokines may be both fused in any order to the N- or C-terminus of theFc region, or one cytokine may be fused at the N-terminus and the otherat the C-terminus.

Some of these permutations are illustrated in FIG. 2. For example, inthe embodiment shown in FIG. 2A, a fusion protein 44 of the invention isfused to the C-terminus of an Fc region containing hinge region 38, CH2region 40 and CH3 region 42. Fusion protein 44 could have a variety ofstructures, including, for example, the structures of fusion proteins10, 16, 24, 26, 28, 30, 32, 34, or 36 depicted in FIGS. 1A–1I. If fusionprotein 44 has more than one N-terminus and C-terminus, as in fusionproteins 16, 24, 26, and 28, the Fc region could be fused to eitherN-terminus of fusion protein 44. As shown in FIG. 2B, fusion protein 44could be fused to the N-terminus of an Fc region. In the embodimentshown in FIG. 2C, a first cytokine 12 could be fused to the N-terminusof an Fc region, and a second cytokine 14 could be fused to theC-terminus of the Fc region.

Structural Considerations

It is important to note that cytokines, as a class of proteins, aresimilar in size and in general folding properties. Thus, the specificexamples disclosed herein illustrate how to construct multiple cytokinefusion proteins for the family of cytokine proteins. For example, manycytokines fall into a protein folding class termed the “four-helixbundle”. Four helix bundle proteins include granulocyte-colonystimulating factor (G-CSF), interleukin 6 (IL-6), leukemia inhibitoryfactor (LIF), growth hormone, ciliary neurotrophic factor (CNTF),leptin, erythropoietin, granulocyte-macrophage colony stimulating factor(GM-CSF), interleukin-5 (IL-5), macrophage colony-stimulating factor(M-CSF), IL-2, IL-4, interleukin-3 (IL-3), IL-10, interferon-beta, theinterferon alphas and the closely related inteferon tau, and interferongamma (IFN-gamma).

With the exception of IL-5 and IFN-gamma, all of these proteins fold asmonomers with four roughly parallel alpha helices and two crossoverconnections. In each case except IL-5 and IFN-gamma, the N-terminus andthe C-terminus are on the same face of the protein. Because thefour-helix bundle proteins, except IL-5 and IFN-gamma, have the samefolding pattern, the methods described herein for IL-2, IL-4 and GM-CSFalso apply to other four-helix bundle proteins and other small cytokineproteins that fold as monomers.

Chemokines are a specific class of cytokines that are thought to formextracellular gradients and mediate the chemotaxis of specific classesof immune cells. For example, MCP-1 is a chemoattractant for monocytes,macrophages, and activated T cells; eotaxin is a chemoattractant foreosinophils; and interleukin-8 is a chemoattractant for neutrophils. Inaddition to their chemoattractant function, chemokines, like othercytokines, are able to induce the expression of specific genes inspecific target cells. For example, MCP-1 is thought to induce theexpression of Tissue Factor in vascular smooth muscle cells (Schecter etal., J Biol Chem. [1997] 272:28568–73).

The invention discloses cytokine-cytokine fusions andantibody-cytokine-cytokine fusions in which one or more cytokines is achemokine. The invention also discloses protein constructs with three ormore cytokines, in which one or more cytokine is a chemokine. Forexample, the chemokines IP-10, RANTES, MIP-1 alpha, MIP-1 beta,macrophage chemoattractant proteins, eotaxin, lymphotactin, BLC, can befused to a second cytokine with or without other moieties such as anantibody moiety.

The human genome, for example, encodes at least 50 chemokines. Knownchemokines generally share a similar three-dimensional monomer structureand protein folding pattern. Accordingly, the general types of proteinconstructs and construction strategies disclosed here can be applied toa variety of known or as-yet-undiscovered chemokines.

Chemokines have a distinct folding pattern with three beta-strands andone alpha helix. Chemokines fold as monomers and, in some but not allcases, then dimerize after folding. For all chemokines, the foldingpattern of the monomer subunit is identical and the overall structuresare extremely similar. For example, the three dimensional structures ofInterleukin-8, Platelet factor 4, Melanoma growth stimulating activity(MGSA), Macrophage: inflammatory protein, MIP, RANTES (regulated upon:activation, normal T-cell expressed and secreted), Monocytechemoattractant protein-1 (MCP-1, MCAF), Eotaxin, Monocytechemoattractant protein-3 (MCP-3), Chemokine domain of fractalkine,Neutrophil-activating peptide-2 (NAP-2), Stromal cell-derived factor-1(SDF-1), Macrophage inflammatory protein-2, Chemokine hcc-2 (macrophageinflammatory protein-5), Gro beta, Cytokine-induced neutrophilchemoattractant, and CINC/Gro have been determined by X-raycrystallography and/or NMR methods; all of these structures show thesame fold and are generally similar. Because the chemokines have thesame folding pattern, the methods described herein for lymphotactin alsoapply to other chemokine proteins.

A free N-terminus of a chemokine is often important for its function.Therefore, it is advantageous in some embodiments to construct fusionsin which a second cytokine, antibody moiety, or other protein moiety maybe fused to the C-terminus of the chemokine. To construct a proteincomplex containing two active chemokines, it is useful, for example, tofuse the two different chemokines to the N-termini of an antibody'sheavy and light chains. Some chemokines, such as IL-8, are dimeric underphysiological conditions. For certain applications, it is useful tocoexpress a multiple cytokine antibody fusion, such as anIL-8-antibody-cytokine fusion, along with an unfused IL-8 moiety or anIL-8 moiety with a different fusion partner that does not interact withthe antibody moiety. In this way, the different IL-8 moieties canheterodimerize without spatial constraints or polymerization that mightresult if all IL-8 moieties were fused to an antibody chain. The desiredmultiple cytokine fusion protein can then be separated on the basis ofsize or on the basis of binding to an antibody-binding protein such asStaphylococcus A protein.

For multiple cytokine fusion proteins comprising a chemokine, it is apreferred embodiment that the fusion protein also comprises a localizingfunction, such as an antibody moiety-that binds to an antigen. Withoutwishing to be bound by theory, it is generally thought that body-widedistribution of a chemokine will have no effect or lead to a generaldesensitization of cells toward that chemokine. In addition, it isthought that the chemoattractant function of a chemokine can only bemanifested when there is a concentration differential of the chemokine.

A preferred embodiment is a lymphokine-antibody-interleukin-2 fusionprotein. Another preferred embodiment is one in which both the chemokineand the second cytokine promote a Th1 response. For example, a fusionprotein comprising IP-10 and IL-12 is one highly preferred embodiment.

Extending the Half-Life of Multiple Cytokines with Short SerumHalf-Lives

The invention also describes fusion proteins comprising two cytokines,both with a short serum half-life, fused to a third moiety with a longserum half-life. For example, when stimulation of dendritic cells isdesired, it is useful to combine the activities of IL-4 and GM-CSF(Thurner, J. Immunol. Methods [1999] 223:1–15; Palucka, et al. [1998] J.Immunol. 160:4587–4595). Because both IL-4 and GM-CSF are smallmolecules with short serum half-lives, it is useful to construct afusion protein comprising an Fc region, IL-4, and GM-CSF. The resultingmolecule is a powerful stimulant of dendritic cell proliferation andactivity. Likewise, both IL-4 and GM-CSF could be fused to a targetingcomponent such as an antibody for the purpose of delivering the combinedcytokine activities to the site of cells expressing a predeterminedantigen.

The Fc region, alone or as part of an intact antibody, can conferseveral properties to multiple cytokine fusions that may be advantageousor disadvantageous, depending on the specific application. Theseproperties include dimerization, extension of serum half-life, abilityto fix complement, ability to mediate antibody-dependent cell-mediatedcytotoxicity (ADCC), and binding to Fe receptors. If extension of serumhalf-life is a primary desired feature and immunological properties ofthe Fe region are unimportant or undesirable, it is preferable to use anFe region that is a natural variant or mutant lacking one or moreimmunological properties. For example, if it is desirable to equalizeand extend the serum half-lives of two or more cytokines with shortserum half-lives, it is preferable to construct a multiple cytokinefusion protein comprising an Fc region from human IgG2 or IgG4, whichrespectively have reduced or no affinity for Fe receptors, or to use anFc region carrying a mutation in the Fc receptor binding site. In fact,it has already been shown that fusion of some cytokines to antibodiesincreases the affinity of the fusion protein to Fe receptors and thatthis results in faster rates of clearance in animals. The use of Fcregions with reduced affinity for Fc receptors was shown to greatlyimprove the serum half-life of these molecules (Gillies et al. [1999]Cancer Res. 59:2159–2166). Under some circumstances and depending on thecytokines used, an Fc region that binds to the Fc receptor will resultin internalization of the multiple cytokine fusion protein anddegradation of one or more cytokine moieties.

Targeting

The invention also describes fusion proteins in which two or morecytokines are attached to a protein that is capable of localizing thecytokines to a particular target molecule, cell, or bodily location. Thepreferred molecule with localizing capability is an antibody or a moietycomprising the antigen-binding variable regions of an antibody. However,other localizing molecules, or domains thereof, may be used, such asspecific ligands or receptors, naturally occurring binding proteins,enzymes that bind to particular substrates, artificially generatedpeptides that have been selected for a particular binding or localizingcapability, peptides with distinctive physico-chemical properties thatresult in a targeting capability, proteins that possess a targetingcapability by virtue of binding to another molecule that is targeted, orother types of proteins. In the case of fusing two cytokines to atargeting molecule, a preferred first cytokine is IL-12. When IL-12 isused, a preferred second cytokine is IL-2 or GM-CSF.

In the case of an antibody, there are a large number of ways in whichtwo or more cytokines can be fused, because there are several possiblesites of attachment. For example, an IgG antibody consists of two heavyand two light chains. The two cytokines may be fused to each other andthen fused to an N- or C-terminus of either the heavy or the lightchain. Alternatively, each cytokine may be fused separately to one ofthe N- or C-termini on the antibody molecule.

FIG. 3 illustrates a subset of ways in two cytokines may be fused to anantibody molecule. For example, referring to FIG. 3A, a fusion protein44 of the invention could be fused to the C-terminus of animmunoglobulin heavy chain 46, which is associated with animmunoglobulin light chain 48. As in FIG. 2, fusion protein 44 can havea variety of structures, including, for example, the structures offusion proteins 10, 16, 24, 26, 28, 30, 32, 34, or 36 depicted in FIGS.1A–1I. As shown in FIG. 3B, a fusion protein 44 could be fused to theN-terminus of an immunoglobulin heavy chain 46 associated with animmunoglobulin light chain 48. In the embodiments shown in FIGS. 3C and3D, fusion protein 44 is fused to the N-terminus (FIG. 3C) or theC-terminus (FIG. 3D) of an immunoglobulin light chain 48 associated withan immunoglobulin heavy chain 46. As shown in FIGS. 3E and 3F, a firstcytokine 12 may be fused to an immunoglobulin light chain 48 associatedwith an immunoglobulin heavy chain 46 fused to a second cytokine 14. Thecytokines 12 and 14 may be fused to the N-termini (FIG. 3E) or theC-termini (FIG. 3F) of the immunoglobulin chains. Alternatively, as inFIG. 3G, first cytokine 12 may be fused to the N-terminus of theimmunoglobulin light chain 48 while the second cytokine 14 is fused tothe C-terminus of the immunoglobulin heavy chain 46.

Fusions to Single-Chain Antibodies

It is sometimes convenient to express antibodies as single-chainmolecules. The invention also provides fusion proteins in which two ormore cytokines are fused to a single-chain antibody. This has theadvantage of reducing the number of the DNA constructs used whenexpressing the desired fusion protein, which may be especially useful ingene therapy. In particular, if the cytokines are single-chainmolecules, then fusion of the cytokines to the single-chain antibodywill allow expression of the fusion protein as a single protein chain.

As shown in FIGS. 4A–4C, in some embodiments, cytokines can be fused tothe single-chain antibody at its N-terminus, its C-terminus, or at bothtermini. For example, as shown in FIG. 4A, fusion protein 44 can befused to the C-terminus of single-chain antibody 50 having light chainvariable region 52 and heavy chain variable region 54. As shown in FIG.4B, fusion protein 44 can also be fused to the N-terminus ofsingle-chain antibody 50. In the embodiment shown in FIG. 4C, a firstcytokine 12 is fused to the N-terminus of single-chain antibody 50, anda second cytokine 14 is fused to the C-terminus of single-chain antibody50.

A preferred embodiment comprises a fusion of IL-12 and a second cytokineto the single-chain antibody. A more preferred embodiment comprises IL-2or GM-CSF as the second cytokine.

The constant regions of antibodies have the potential to mediate avariety of effector functions. For example IgG1 mediates complementfixation, ADCC, and binding to Fc receptor. The position at which thecytokine is fused may alter the antibody constant region's effectorfunction, which is useful if modulation of these effector functions isdesired.

In some cases it may be desirable to construct a fusion of two or morecytokines to a moiety with the targeting region of an antibody, butwithout the constant regions. Such a fusion protein is smaller than afusion of a complete antibody to two or more cytokines, which may beadvantageous for certain purposes. In addition, such a fusion proteinwill lack one or more of the effector functions of an intact antibody,but will retain the targeting capability of an antibody.

The invention therefore features fusion proteins in which two or morecytokines are fused to a single-chain Fv region. As shown in theembodiments depicted in FIGS. 5A–5C, two cytokines may be fused to theN-terminus or the C-terminus of the Fv region, or one cytokine to eachterminus. For example, as shown in FIG. 5A, a fusion protein 44 of theinvention may be fused to the C-terminus of a single-chain Fv regioncontaining an immunoglobulin light chain variable region 52 and animmunoglobulin heavy chain variable region 54. A fusion protein 44 mayalso be fused to the N-terminus of an Fv region as shown in FIG. 5B. Asshown in FIG. 5C, a first cytokine 12 may be fused to the N-terminus ofan Fv region, and a second cytokine 14 may be fused to the C-terminus ofthe Fv region.

Antibodies as Heterodimeric Vehicles for Multiple Cytokines

In some circumstances, it is desirable to construct a fusion of two ormore cytokines in which, for two of the cytokines, the same end of theprotein is essential for activity. For example, it may be that thenaturally occurring N-terminus of two different cytokines is essentialfor the activity of each cytokine. It is not possible to construct asingle polypeptide chain fusion protein in which both cytokine moietieswould be active.

Antibodies are heterodimeric proteins consisting of heavy and lightchains that are covalently linked by disulfide bonds. If it is desiredto construct a multiple cytokine fusion protein with two cytokinemoieties that both require an intact, unfused N-terminus, it ispreferable to separately fuse the two cytokines to the N-termini of theheavy and light chains of an antibody (FIG. 3E). Similarly, if it isdesired to construct a multiple cytokine fusion protein with twocytokine moieties that both require an intact, unfused C-terminus, it ispreferable to separately fuse the two cytokines to the C-termini of theheavy and light chains of an antibody (FIG. 3F). If the antibody is usedsolely as a vehicle to connect two cytokines in this manner, it may beuseful to mutate or delete those portions of the antibody that conferadditional properties related to immune function. For example, it may bepreferable to use an Fab region as a vehicle, since the Fab regionretains the heterodimerization feature of an antibody but lacks thefunctions characteristic of the Fc region. It may also be useful to usean antibody or antibody fragment in which the antigen combining site isnon-functional.

Fusions of multiple cytokines to antibodies combine many of the novelfeatures of the invention. In antibody-multiple cytokine fusions, theserum half-life of the cytokines is equalized and extended; the activityof both cytokines is localized to a target and the especially toxiceffects due to systemic administration of multiple, synergisticallyacting cytokines are avoided; each cytokine is effectively dimerized ormultimerized; and the cytokines do not need to be directly fused but maybe fused to different sites on the heavy and light chains of theantibody molecule.

In designing a fusion protein comprising multiple cytokines and anantibody, there are a number of options and configurations that can bedistinguished by routine experimentation. Structural biologyconsiderations are also useful. For example, many cytokines fall into aclass termed 4-helix bundles. These structures consist of four alphahelices and have the N-terminus and C-terminus in the same vicinity. Ingeneral, the face of a cytokine around the N- and C-terminus is not usedin binding to a cytokine receptor, so either terminus can be used forfusion to and antibody or to a second cytokine. However, it is sometimesdifficult to directly fuse both the N- and C-terminus of a 4-helixbundle cytokine to different moieties, for steric reasons. When it isdesirable to fuse two different 4-helix bundle cytokines to an antibody,it is therefore useful to fuse each cytokine to a different site on theantibody. Alternatively, if it is necessary to construct a polypeptidechain of the form Ig chain-cytokine-cytokine, one or more flexiblelinkers may be used to overcome the steric problems.

Instead of an antibody, it is also possible to use other secretedheterodimeric molecules to carry multiple cytokines. For example, acomplex including prostate-specific antigen and the protease inhibitorwith which it complexes, the IgA heavy chain and the J chain, members ofthe TGF-beta family and their astacin-like binding partners, or IL-12could be used.

Nucleic Acids

The invention also features nucleic acids capable of expressing each ofthe above types of proteins. These include nucleic acids encoding fusionproteins comprising two or more cytokines, fusions comprising two ormore cytokines and a dimerization domain such as an Fc region, fusionscomprising two or more cytokines fused to an antibody, and two or morecytokines fused to an Fv region. Preferred forms of the nucleic acidsare DNA vectors from which the fusion proteins can be expressed ineither bacteria or mammalian cells. For fusion proteins that comprisemultiple polypeptide chains, more than one encoding nucleic acid may beused. Alternatively, it may be useful to place two or more fusionprotein coding sequences on a single nucleic acid molecule. The Examplesillustrate particular forms of the featured nucleic acids encodingmultiple cytokines.

The nucleic acids of the invention are particularly useful forexpression of multiple cytokine fusion proteins, for either theproduction of these proteins or for gene therapy purposes.

Methods for synthesizing useful embodiments of the invention, as well asassays useful for testing their pharmacological activities, aredescribed in the Examples.

The present invention also provides pharmaceutical compositions andmethods of their use in treatment and prevention of a wide variety ofdiseases, including but not limited to treatment of various infectionsand cancer, and vaccination against various diseases.

Multiple cytokine fusion proteins can be used to treat bacterial,parasitic, fungal, or viral infections, or cancer. For example, IL-12 isknown to have a protective effect in many types of infections, includingbut not limited to infections with the bacterium. Listeriamonocytogenes; the parasites Toxoplasma gondii, Leishmania major, andSchistosoma mansoni; the fungus Candida albicans; and the viruseschoriomeningitis virus and cytomegalovirus. Since cytokines generallyact in combination, it is often useful to use fusion proteins comprisingtwo or more cytokines that are known to act synergistically. Forexample, since IL-2 potentiates the effects of IL-12, it is useful tocombine these cytokines in treatment of bacterial, parasitic, fungal andviral diseases.

A preferred method of treatment of infectious disease is to use multiplecytokine fusion proteins that are further fused to a targeting agentthat places the multiple cytokine at the site of infection. Varioustargeting strategies are described below.

The pharmaceutical compositions of the invention may be used in the formof solid, semisolid, or liquid dosage forms, such as, for example,pills, capsules, powders, liquids, suspensions, or the like, preferablyin unit dosage forms suitable for administration of precise dosages. Thecompositions will include a conventional pharmaceutical carrier orexcipient and, in addition, may include other medicinal agents,pharmaceuticals agents, carriers, adjuvants, etc. Such excipients mayinclude other proteins, such as, for example, human serum albumin orplasma proteins. Actual methods of preparing such dosage forms are knownor will be apparent to those skilled in the art. The composition orformulation to be administered will, in any event, contain a quantity ofthe active component(s) in an amount effective to achieve the desiredeffect in the subject being treated.

Administration of the compositions hereof can be via any of the acceptedmodes of administration for agents that exhibit such activity. Thesemethods include oral, parenteral, or topical administration andotherwise systemic forms. Injection is a preferred method ofadministration.

The amount of active compound administered will, of course, be dependenton the subject being treated, the severity of the affliction, the mannerof administration, and the judgment of the prescribing physician.

As described above, cytokines such as IL-2, IL-12, GM-CSF, IL-4, andothers have been investigated for treatment of cancer. Under somecircumstances it is advantageous to use a multiple cytokine fusionprotein in treatment of cancer, for reasons of simpler administration,increased serum half-life of one of the component cytokines, and/orsuperior modulation of the relative activities of the two cytokines.

A preferred method of treatment of cancer is to target the cytokines toa particular organ or tissue, so the effect of the cytokines may beconcentrated and the side effects of systemic distribution may beavoided. For example, fusions of multiple cytokines to an Fe region areexpected to be concentrated to the liver, which may be useful intreatment of cancer limited to the liver. A more preferred method is touse a multiple cytokine fusion protein that is further fused to atargeting agent such as an antibody. In particular, the antibodiesKS-1/4 and 14.18 are directed against tumor-specific antigens (Varki N Met al., Cancer Res [1984] 44:681–7; Gillies et al., Journal ofImmunological Methods 125:191 [1989]; U.S. Pat. Nos. 4,975,369 and5,650,150). When using antibody-multiple cytokine fusion, it is oftenuseful to investigate the type of tumor and choose an antibody directedagainst an antigen that is likely to be present on that type of tumor.For example, it may be useful to characterize the tumor by FACSanalysis, Western blot, examination of the tumor's DNA, or simplyidentifying the type of the tumor cell. Such methods of tumorcharacterization are well known to those skilled in the art of tumorcharacterization, such as oncologists and tumor biologists. It is alsopossible to target multiple cytokine fusion proteins by a variety ofother means, such as fusion to specific ligands or receptor moieties,fusion to peptide aptamers with pre-selected binding activities,chemical conjugation to small molecules with localizing characteristics,and so on. These targeting methods may also be used for treatment ofother diseases, such as infections.

Treatment of Cancer and Other Cellular Disorders by Gene Therapy

The nucleic acids of the invention may be used as gene therapy agentsfor treatment of cancer and other diseases in which it is desirable totarget the immune system to a specific cell type. For example, cancercells are withdrawn from a human or animal, one or more nucleic acidsencoding a multiple cytokine fusion protein are transfected into thecancer cells, and the cancer cells are then reintroduced into the humanor animal. Alternatively, the DNA may be introduced into the cancer cellin situ. The human or animal then mounts an immune response to thecancer cells, which may cure or lessen the severity of the cancer. Amultiple cytokine gene fusion, coupled to appropriate regulatoryelements to promote expression in mammalian cells, may be transfectedinto the cancer cells by any of a variety of techniques, include thecalcium phosphate method, a ‘gene gun’, adenovirus vectors, cationicliposomes, retroviral vectors, or any other efficient transfectionmethod. The nucleic acid may encode a multiple cytokine fusion proteinthat is further fused to other moieties.

Anti-cancer gene therapy with a nucleic acid expressing a fusion of morethan one fused cytokine, may be combined with other cancer treatments,such as treatments that may augment the immune-stimulating properties ofthe fused cytokine protein. For example, the nucleic acid of theinvention may also express other protein moieties that may aid in thedevelopment of an immune response to antigens expressed by the cancercells, or may be co-transfected with other nucleic acids expressing suchprotein moieties. In particular, nucleic acids expressing the B7costimulatory surface protein may be cotransfected into the cancer cells[Robinson et al., U.S. Pat. No. 5,738,852]. Transfection of cancer cellswith a nucleic acid expressing multiple cytokine fusion may also beaccompanied by treatment with an antibody or immunocytokine that targetsthe cancer cells [Lode et al. (1998) Proc. Nat. Acad. Sci. 95:2475].Transfection of cancer cells with a nucleic acid expressing multiplecytokine fusion may also be accompanied by treatment with anangiogenesis blocker [Lode et al. (1999) Proc. Nat. Acad. Sci. 9:1591].

Therapies using additional immune stimulators and/or angiogenesisblockers may also be combined with systemic treatment with multiplecytokine fusion proteins. An advantage of co-treatment with additionalimmune stimulators or angiogenesis blockers is that these treatments,unlike DNA-damaging agents and cell-cycle blockers, do not kill immunecells that may be dividing due to stimulation by the multiple cytokinefusion protein.

A preferred embodiment of this gene therapy method is to introduce oneor more nucleic acids encoding IL-12 and a second cytokine into cancercells, and then reintroduce the cancer cells into the humor animal. Thesecond cytokine is preferably. IL-2 or GM-CSF.

The present invention provides novel vaccine compositions and methods ofadjuvantation of vaccines intended to provide a protective cell-mediatedimmune response in vaccinated host mammals against certain pathogens,using as an adjuvant two or more cytokines that have been fused. Forexample, if a Th1 immune response is desired, multiple Th1-promotingcytokines may be fused and the resulting fusion protein administered toan animal in combination with an antigen.

In particular, IL-12 and IL-2 may be fused and administered with anantigen. Alternatively, IL-12 and IL-2 may be further fused to anantigenic protein itself and used to stimulate an immune response. Inthis case, the invention is directed to vaccines that rely on the host'scell-mediated immunity, i.e. the elicitation of cytotoxic T lymphocytesand activated phagocytes to provide protection against infection by aparticular pathogen. It is especially useful to perform vaccinationswith fusion proteins comprising IL-12, IL-2, and the antigen, as thiscombination directs a Th1 response against the antigen. Conventionaladjuvants used in humans, such as alum, tend to induce a Th2 response.

If a Th2 immune response is desired, fused combinations of Th2-promotingcytokines may be used. For example, it may be useful to fuse IL-4 andIL-10 to form a single molecule, and the resulting fusion protein usedas an adjuvant. In particular, if it is desired to recruit dendriticcells in an animal, fused combinations of IL-4 and GM-CSF may be furtherfused with either an Fc region to promote binding to antigen-presentingcells, or further fused to an antibody capable of directing the fusedcytokines to a target tissue such as a tumor.

The present invention also provides novel therapeutic compositions andmethods of adjuvanting intended to provide a synergistic effect withcertain therapeutic compositions, including so-called ‘cancer vaccines,’which may include a selected antigen occurring on a cancer cell. Forexample, a protein comprising two or more fused cytokines may beadministered directly by an appropriate route, along with appropriatelytreated cancer cells.

EXAMPLES Example 1 Construction of Gene Fusions Capable of ExpressingCytokine-Cytokine Fusion Proteins

To create multifunctional proteins having a plurality of cytokines, genefusions between IL-12's p40 and IL-2, and between IL-12's p40 and GM-CSFwere synthesized. In addition, the coding sequence for mature murine p35(SEQ ID NO:1) was fused to a promoter and leader sequence that allowhigh levels of expression and efficient secretion. Coding sequences ofmurine p40-IL-2 and p40-GM-CSF are shown in SEQ ID NO:2 and SEQ ID NO:3,respectively. A human p40-IL-2 fusion was also constructed (SEQ IDNO:4). Fusions of a mouse Fc region of IgG2a to mouse p35 (SEQ ID NO:5)and of human p35 to a human Fc region of IgG1 (SEQ ID NO:6) wereconstructed, using expression plasmids previously disclosed (Lo et al.Protein Engineering 11:495–500 [1998]; Lo et al., U.S. Pat. No.5,726,087).

Fusion of mature mouse and human p35 to the C-terminus of the KS-1/4antibody heavy chain is described by Gillies et al. (J. Immunology[1998] 160:6195–6203). Fusions of mature mouse and human p35 to theC-terminus of the 14.18 antibody heavy chain were constructed in ananalogous manner (PCT International Publication WO99/29732).

The type of strategy discussed here to fuse p40 to IL-2 and to GM-CSF isgenerally applicable to the fusion of two or more cytokines.Specifically, the coding sequence of the most N-terminal moietycomprises a signal sequence for secretion, while the C-terminal moietiesdoes not require a signal sequence. In some circumstances, it may beuseful to place a coding sequence for a short peptide linker, preferably10–15 amino acids long and rich in glycine and serine, between thecoding sequences for the two cytokines. The DNA manipulations involvedin generating all such types of fusions are within the level of skill inthe art.

For example, details of the construction of a fusion between the murineIL-12 p40 subunit and murine IL-2 were as follows. Full length cDNA ofthe p40 subunit of murine IL-12 was cloned by PCR from mouse spleencells activated with Concavalin A (5 μg/ml in culture medium for 3days). The forward primer had the sequence AA GCTAGC ACC ATG TGT CCT CAGAAG CTA ACC (SEQ ID NO:7), in which a NheI site GCTAGC (residues 3–8 ofSEQ ID NO:7) was placed upstream of the translation initiation codonATG, and the reverse primer had the sequence CTC GAG CTA GGA TCG GAC CCTGCA GGG (SEQ ID NO:8), in which an XhoI site CTCGAC; (residues 1–6 ofSEQ ID NO:8) was placed immediately downstream of the translation stopcodon TAG (anticodon CTA). After sequence verification, the NheI-XhoIfragment containing the mu-p40 cDNA with its native leader was ligatedto the XbaI-XhoI digested expression vector pdCs [Lo et al. (1998)Protein Engineering 11:495–500]. The restriction sites NheI and XbaIhave compatible sticky ends, and NheI site was used for the cloning ofmu-p40 because mu-p40 has an internal XbaI site.

For the construction of DNA encoding mu-p40-muIL-2, an oligonucleotidelinker was used to join the mu-p40 DNA via its PstI site (C TGC AG) to aSmaI-XhoI fragment containing the cDNA of mature murine IL-2. The DNAsequence at the junction of the fusion protein was C TGC AGG GTC CGA TCCCCG GGT AAA GCA CCC (SEQ ID NO:9), where C TGC AG (residues 1–6 of SEQID NO:9) is the PstI site, C CCG GG (residues 15–20 of SEQ ID NO:9) isthe SmaI site, TCC is the C-terminal amino acid residue of murine p40,and GCA is the N-terminal residue of mature murine IL-2.

The DNA encoding single-chain muIL12-muGMCSF was derived from the DNAconstruct encoding single-chain muIL12-muIL2 above by replacing themuIL2 cDNA by the muGMCSF cDNA at the SmaI site. The DNA sequence at thejunction of single-chain muIL12 and muGMCSF was C TGC AGG GTC CGA TCCCCG GGA AAA GCA (SEQ ID NO:10), where C TGC AG (residues 1–6 of SEQ IDNO:10) is the PstI site, C CCG GG (residues 17–22 of SEQ ID NO:10) isthe SmaI site, TCC is the C-terminal amino acid residue of murine p40,and GCA is the N-terminal residue of mature murine GMCSF.

Example 2 Expression of IL-12 Fusion Proteins

IL-12-IL-2 fusion proteins were expressed as follows. Differentcombinations of the individual vectors encoding p40 fusions and vectorsencoding proteins comprising p35 were co-transfected into human 293epidermal Carcinoma cells for transient expression of fusion proteins.DNA was purified using preparative kits (Wizard, Promega Inc.),ethanol-precipitated for sterilization and resuspended in sterile water.

For expression of biologically active IL-12 fusion protein heterodimers,different combinations of the individual vectors encoding fusion andnon-fusion forms of the subunits were transiently expressed byco-transfection of human 293 epidermal carcinoma cells. DNA was purifiedusing preparative kits (Wizard, Promega Inc.), ethanol precipitated forsterilization and resuspension in sterile water. Calcium phosphateprecipitates were prepared by standard methods using 10 μg of DNA per ml(5 μg of each when two plasmids were co-transfected) and 0.5 ml/platewere added to cultures of 293 growing in 60 mm plates at approximately70% confluency (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor LaboratoryPress, 1989). After 16 hr, the medium containing the precipitate wasremoved and replaced with fresh medium. After 3 days, the supernatantwas removed and analyzed for production of transfected gene expressionby ELISA, biological determination of IL-12 activity, orimmunoprecipitation and analysis on SDS gels of radioactively labeledproteins. For labeling, medium without methionine was used to replacethe growth medium on the second day of culture and ³⁵S-methionine (100μCi/ml) was added. After an additional 16 hr incubation, the media washarvested, clarified by centrifugation (5 ml at 13,000 rpm in a tabletop microcentrifuge) and incubated with protein A Sepharose beads (10 μlof bead volume per ml of culture supernatant). After 1 hr at roomtemperature, the beads were washed by repeated centrifugation andresuspension in PBS buffer containing 1% Nonidet—P40 (NP-40). The finalpellet was resuspended in SDS-containing gel buffer and boiled for 2min. After removing the beads by centrifugation, the supernatant wasdivided into two aliquots. Reducing agent (5% 2-mercaptoethanol) wasadded to one sample and both samples were boiled for 5 min prior toloading on an SDS polyacrylamide gel. After electrophoresis the gel wasexposed to X-ray film (autoradiography).

Transfections using the following expression plasmids were performed:mu.p35 plus mu.p40-IL-2, KS-1/4-mu.p35 plus mu.p40, KS-1/4-mu.p35 plusmu.p40-IL-2, 14.18-mu.p35 plus mu.p40-IL-2, hu.Fc-.p35 plus hu.p40-IL-2,KS-1/4-hu.p35 plus hu.p40-IL-2, and 14.18-hu.p35 plus hu.p40′-IL-2,where “mu” refers to murine proteins and “hu” refers to human proteins.

When cells were metabolically labeled with ³⁵S-methionine and secretedproteins examined by reducing SDS gel electrophoresis andautoradiography, high level expression was observed in each case.Molecular weights of reduced fusion proteins were predicted based on themolecular weights of component proteins, as follows: p35 of IL-12, 35kD; p40 of IL-12, 40 kD; IL-2, 16 kD; Fc, 32 kD; Ig heavy chain, 55 kD;and Ig light chain, 28 kD. Proteins migrating with approximately thepredicted molecular weights were observed.

Stably transfected cell lines expressing multiple cytokine fusionproteins were also isolated. In the case of heterodimeric constructs,IL-12 p40-IL-2 or IL-12 p40-GM-CSF fusion protein encoding expressionvectors as described earlier for the IL-12 p40 subunit alone (Gillies etal. [1998] J. Immunol. 160: 6195–62030). Transfected cell linesexpressing the p40 fusion proteins were transfected a second time withexpression vectors encoding either the IL-12 p35 subunit, an Fc-p35fusion protein or an antibody-p35 fusion protein expression vector asdescribed (Gillies et al. [1998] J. Immunol. 160: 6195–62030).

Supernatant from stably transfected cells expressing human FC-IL-12-IL-2(i.e. expressing KS-p35 and p40-IL-2) was collected and the productswere purified by binding to and elution from protein A Sepharoseaccording to the manufacturer's procedures (Repligen, Needham, Mass.).The pure proteins were assayed by ELISA for IL-12 and IL-2 content. Theresults showed that the individual cytokine contents were approximately4-fold different by mass which correlates to the 4-fold difference inmolecular weight between IL-12 and IL-2. Similarly, assay of theproducts of transfected cells expressing human KS-IL-12-IL-2 by ELISAfor IL-12 and IL-2 levels gave similar values of IL-12 and IL-2. Thus,within the accuracy of the ELISAs, the measured values indicate thatIL-12 and IL-2 are being produced in about a 1:1 molar ratio. The sameresults were obtained with the IL-12-GM-CSF fusion proteins made eitherwith the Fe or whole antibody.

Example 3 Synergistic Activity of Fusion Proteins in an IFN-γ InductionAssay

Biological activity of IL-12-IL-2 fusion proteins was measured in anIFN-γ induction assay using either resting or mitogen-activated humanperipheral blood mononuclear cells (PBMCs) from human volunteers (FIG.6). IFN-γ production was measured by ELISA.

Human peripheral blood mononuclear cells (PBMCs) were obtained fromhealthy volunteers and were purified by centrifugation on aFicoll-Hypaque (Pharmacia) gradient (1700 rpm for 20 min). The “buffy”coat containing the PBMC was diluted with serum-free culture medium(SF-RPMI) to a volume of 50 ml and collected by centrifugation at 1500rpm for 5 min. After gradient centrifugation, cells were resuspended incell culture medium containing 10% fetal bovine serum (RPMI-10) with orwithout phytohemagglutinin (PHA; 10 μg/ml) at a density of 5×10⁶cells/ml and were cultured for 3 days at 37° C. in a humidified CO₂incubator. The cells were collected by centrifugation, washed threetimes with an equal volume of SF-RPMI and resuspended in fresh RPMI-10(1×10⁶ cells/ml). Aliquots (100 μl) were dispensed into the wells ofmultiple 96-well plates to give a final cell number of 10⁵ per well.Test samples from culture medium were serially diluted in fresh culturemedium and added to wells of the 96-well plate. Control wells receivedIL-12 (FIG. 6A) or an equimolar mixture of commercial IL-2 and IL-12(FIG. 6B; cytokines purchased from R & D Systems). The plates wereincubated for 48 hr at 37° C. in a CO₂ incubator at which time aliquots(20 μl) were removed for analysis of IFN-γ concentration by ELISA,following the instructions of the manufacturer (Endogen, Inc., Woburn,Mass. USA).

In FIG. 6A, the activities of the human IL-12-IL-2 fusion protein werecompared with IL-12 alone. The results illustrate that IL-12 aloneinduced IFN-γ to moderate levels, while the IL-12-IL-2 fusion proteinstrongly induced IFN-γ synthesis. Since IL-2 is also known to beinsufficient for IFN-γ synthesis, these results indicate that the IL-12and IL-2 moieties are both functional within the fusion protein andfunction synergistically.

Next, the activities of an Fc-IL-12-IL-2 fusion protein, a KS-IL-12-IL-2fusion protein, and a mixture consisting of 1:1 molar ratio of IL-12 toIL-2 were compared for their ability to induce IFN-γ. The results inFIG. 6B indicate that the Fc-IL-12-IL-2 fusion protein and KS-IL-12-IL-2fusion protein have about the same activity as an equimolar mixture ofIL-12 and IL-2. The same results were obtained when the mouse forms ofIL-2 and IL-12, constructed in the manner just described in Example 1for the human forms, were used for the construction of fusion proteins.

Example 4 IL-2 and IL-12 Bioactivity of IL-12-IL-2 Fusion Proteins

The activities of IL-2 and IL-12 in the fusion proteins were compared tothe free cytokines in proliferation-based assays. The activity of amurine antibody 14.18-IL-12-IL-2 molecule was tested in a typical IL-12proliferation assay. Human PBMCs were obtained from volunteers andcultured with 5 micrograms/ml of phytohemagglutinin-P for three days,washed with Hank's HBSS, and plated into microtiter plates at 10⁵ cellsper well, according to a standard procedure (Gately, M. K., Chizzonite,R., and Presky, D. H. Current Protocols in Immunology [1995] pp.6.16.1–6.16.15). Cells were incubated in the presence of various testproteins for 48 hours, and 0.3 microCuries of ³H-thymidine were addedten hours before determining levels of radioactive incorporation. IL-12and an equimolar mixture of IL-12 and IL-2 stimulated incorporation of³H-thymidine into cells in a dose-dependent manner, and the14.18-IL-12-IL-2 fusion protein was about equally effective instimulating incorporation of 3H-thymidine. IL-2 stimulated incorporationof ³H-thymidine only at higher molar concentrations, indicating that theobserved incorporation of ³H-thymidine stimulated by the14.18-IL-12-IL-2 fusion protein is due primarily to its IL-12 activity.Results are shown in FIG. 7.

In addition, the biological activity of the IL-2 moiety was tested in adifferent cell proliferation assay, following a standard procedure(Davis, L. S., Lipsky, P. E., and Bottomly, K. Current Protocols inMolecular Immunology [1995] p. 6.3.1–6.3.7). The mouse CTLL-2 cell linedepends on IL-2 for proliferation. The CTLL-2 cell line can alsoproliferate in response to IL-4, but is not responsive to IL-12. CTLL-2cells in active log-phase growth were washed twice in medium lackingIL-2 and plated at about 1×10⁴ cells per well in microtiter wells in thepresence of various amounts of commercial murine IL-2, murine14.18-IL-12-IL-2 fusion protein, or commercial murine IL-12, and grownfor 48 hours. At the end of the growth period, the number of viablecells was quantitated using the MTT/MTS assay. FIG. 8 shows anexperiment in which levels of IL-2, IL-12, or 14.18-IL-12-IL-2 fusionprotein were varied. The results indicate that murine IL-2 and murine14.18-IL-12-IL-2 fusion protein are about equally potent in stimulatingproliferation, while increasing amounts of murine IL-12 caused nodetectable stimulation of cell proliferation. This result indicates thatthe stimulation of CTLL-2 cell proliferation by 14.18-IL-12-IL-2 fusionprotein is due to the IL-2 moiety and not the IL-12 moiety.

Example 5 Construction and Expression of Single-Chain and Multiple ChainIL-12-IL-2 Fusion Proteins with and Without Antibody Moieties

A single-chain murine IL-12-IL-2 fusion protein was constructed asfollows. A p40-IL-2 coding sequence fusion was constructed by methodsanalogous to those used in construction of the human p40-IL-2 fusion inexample 1. To connect the DNAs encoding the p35 and p40 subunits ofIL-12 and generate a single coding sequence, a DNA encoding a linker wassynthesized with an XhoI site at the 5′ end and a BamHI site at the 3′end. The 5′ end of the mature p40-IL-2 coding sequence was modified tointroduce a restriction site, and then ligated to the 3′ end of thelinker. The 3′ end of the murine p35 coding sequence was modified togenerate a restriction site and ligated to the XhoI site of the linker.The cDNAs encoding single-chain muIL12 and mu-p40-muIL2, described inExample 1, were combined by using a convenient restriction site in p40,to give a third DNA construct encoding single-chain muIL12-muIL2. Thesesteps were carried out using various vectors and DNA fragment isolationsas needed. The sequence of the resulting murine p35-linker-p40-IL-2coding region is SEQ ID NO:11.

At the same time, a corresponding single-chain murine IL-12 codingsequence was constructed by corresponding methods. The coding sequenceis SEQ ID NO:12.

In addition, we further constructed a DNA sequence that encodes a murineIgG2a Fc region fused to the N-terminus of p35-linker-p40-IL-2. Thecoding sequence is SEQ ID NO:13.

Cultured 293 cells were transfected with expression plasmids encodingthe murine single-chain Fc-IL-12-IL-2 and Fc-IL-12 proteins. Expressionof fusion proteins was assayed as described in Example 2. Fc fusionproteins were purified by their binding to protein A Sepharose, and highlevels of expression of Fc-IL-12-IL-2 and Fc-IL-12 were observed.Proteins were synthesized intact, as inferred by the apparent molecularweights from migration on SDS gels: Fc-IL-12-IL-2, 123 kD; and Fc-IL-12,107 kD.

The KS-scIL12-IL2 fusion protein described in Example 1 is a tetramerwith two different polypeptide chains: the KS-1/4 light chain and theKS-1/4 heavy chain with the scIL12-IL2 moiety at the C-terminus. Toinvestigate which sites on an antibody molecule are suitable forattachment of cytokine moieties, a second fusion protein was constructedin which the KS-1/4 antibody, IL-12, and IL-2 moieties were in aconfiguration distinct from the KS-IL12-IL2 configuration in Example 1.This second protein was tetrameric and consisted of two differentpolypeptides. One polypeptide consisted of the light chain of the KS-1/4antibody. The other polypeptide consists of a single-chain muIL12 fusedto the mature N-terminus of the heavy chain of the KS1/4 antibody,followed by murine IL-2 at the carboxyl terminus of the heavy chain.

The cDNA encoding the p35 subunit of murine IL-12 was cloned by PCR frommouse spleen cells activated with Concanavalin A (5 μg/ml in culturemedium for 3 days). The forward primer has the sequence AAGCTT GCTAGCAGCATG TGT CAA TCA CGC TAC (SEQ ID NO:14), where a HindIII site AAGCTT(residues 1–6 of SEQ ID NO:14) is placed upstream of the translationinitiation codon ATG, and the reverse primer has the sequence CTCGAG CTTTCA GGC GGA GCT CAG ATA GCC (SEQ ID NO:15), where an XhoI site CTCGAG(residues 1–6 of SEQ ID NO:15) is placed downstream of the translationstop codon TGA (anticodon TCA).

The DNA encoding the single-chain IL-12 comprises of the mup35 DNAjoined to oligonucleotides encoding a linker rich in glycine and serineresidues, followed by mup40 DNA. The resultant construct has thefollowing sequence at the oligonucleotide junction:

              Ser Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly GlySer Ala (SEQ ID NO:17) G AGC TCC GCG TCG AGC GGG GGC AGC GGG GGC GGA GGCAGC GGC GGG GGC GGA TCC GCC ATG (SEQ ID NO:16)where G AGC TC (residues 1–6 of SEQ ID NO:16) is a SacI restriction sitejust upstream of the murine p35 translation stop codon, GCG encodes theC-terminal amino acid residue of murine p35, GGA TCC (residues 50–55 ofSEQ ID NO:16) is a BamHI restriction site introduced to facilitateligation, and ATG encodes the N-terminal residue of mature mu-p40.

The DNA encoding single-chain muIL12-KS Heavy chain-muGMCSF has thefollowing sequence at the junction of mup40 and the mature N-terminus ofKS heavy chain:

                      Pro Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser GlyGly (SEQ ID NO:19) C TGC AGG GTC CGA TCC CCG GGA TCC GGA GGT TCA GGG GGCGGA GGT AGC GGC GGA (SEQ ID NO:18) Gly Gly Ser Leu Ser GGG GGC TCC TTAAGC CAGwhere C TGC AG (residues 1–6 of SEQ ID NO:18) is a PstI site justupstream of the murine p40 translation stop codon, TCC encodes theC-terminal amino acid residue of murine p40, and CAG encodes theN-terminal residue of mature KS heavy chain. The resultant DNA encodingsingle-chain muIL12-KS Heavy chain-muIL2 was then coexpressed with theKS light chain.

To further investigate which ends of an antibody molecule are availablefor the generation of fusion junctions and to investigate how manydistinct polypeptides can be assembled into a multiple cytokine fusionprotein, a third protein containing KS-1/4, IL-12, and IL-2, namelyIL12-KS(Light chain)+KS(heavy chain)-IL2, was expressed and tested foractivity. This fusion protein is hexameric and comprises three differentpolypeptides. One polypeptide consists of the murine p35 fused to thelight chain of the KS1/4 antibody. A second polypeptide consists of theheavy chain of the KS1/4 antibody fused to human IL-2 [Gillies et al.(1992) Proc. Natl. Acad. Sci. 89:1428], and a third polypeptide is themurine p40. Upon expression, two light chains and two heavy chains aredisulphide bonded to form the tetrameric antibody-cytokine structure. Inaddition, the p35 at the N-terminus of the light chain also isdisulphide bonded with the p40.

The DNA encoding the mup35-KS light chain has the following sequence atthe junction:

              Ser Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly GlySer Leu Ser (SEQ ID NO:21) G AGC TCC GCG TCG AGC GGG GGC AGC GGG GGC GGAGGC AGC GGC GGG GGC GGA TCC TTA AGC GAG (SEQ ID NO:20)where G AGC TC (residues 1–6 of SEQ ID NO:20) is a SacI restriction sitejust upstream of the murine p35 translation stop codon, GCG encodes theC-terminal amino acid residue of murine p35, GGA TCC (residues 50–55 ofSEQ ID NO:20) is a BamHI restriction site introduced to facilitateligation, and GAG encodes the N-terminal amino acid residue of the lightchain.

For expression of this hexameric fusion protein, a murine p40-expressingcell line was generated by transfection with an expression vectorcontaining a neomycin resistance gene and selection by G418. The murinep40-expressing cell line was then transfected with an expression vectorcontaining both the light chain and heavy chain transcription units anda dihydrofolate reductase selection marker, which allowed selection bymethotrexate [Gillies et al. (1998) J. Immunol. 160:6195].

Example 6 Activity of Murine Single-Chain IL-12-IL-2 Fusion Proteins

The same methods used in Example 4 were used to test the activity ofmurine single-chain IL-12-IL-2 produced by transient expression. Theamount of each cytokine in the cell culture supernatant was firstdetermined by ELISA and used to set up a dose-response curve. Theactivities closely corresponded to what was found with the Fe andantibody IL-12-IL-2 fusion proteins and described above.

Specifically, the IL-12 activity of a murine single-chain (sc)IL-12-IL-2 and murine Fc-scIL-12-IL-2 molecules were tested in the humanPBMC cell proliferation assay described in Example 4. IL-12 and anequimolar mixture of IL-12 and IL-2 stimulated incorporation of³H-thymidine into cells in a dose-dependent manner. On a per mole basis,both scIL-12-IL-2 and Fc-scIL-12-IL-2 fusion proteins were about aseffective as IL-12 in stimulating incorporation of ³H-thymidine (FIG.9). As described in Example 4, IL-2 stimulates incorporation of³H-thymidine only at a much higher molar concentrations, indicating thatthe observed incorporation of ³H-thymidine stimulated by thescIL-12-IL-2 fusion proteins is due primarily to their IL-12 activity.

In addition, the biological activity of the IL-2 moiety in thescIL-12-IL-2 fusion proteins was tested in a cell based assay, and wasfound to be about the same as commercial IL-2 on a per mole basis, towithin the accuracy of the assay. The biological activity of the IL-2moiety was tested in the CTLL-2 cell proliferation assay, as describedin Example 4. The results indicate that murine IL-2, murinescIL-12-IL-2, and murine Fc-IL-12-IL-2 fusion protein were about equallypotent in stimulating proliferation. Murine IL-12 causes no detectablestimulation of CTLL-2 cell proliferation. These results indicate thatthe stimulation of CTLL-2 cell proliferation by scIL-12-IL-2 fusionproteins was due to the IL-2 moiety and not the IL-112 moiety.

The IL-12 and IL-2 activities of the Fc-IL12-IL2, IL12-KS-IL2, andIL12-KS(Light chain)+KS(Heavy chain)-IL2 proteins described in Example 5were also tested in cell-based assays. Using the PBMC cellproliferation/tritiated thymidine incorporation assay, the Fc-IL12-IL2,IL12-KS-IL2, and IL12-KS(Light chain)+KS(Heavy chain)-IL2 proteins allshowed potent IL-12 activity. Similarly, using the CTLL-2 cellproliferation assay, the Fc-IL12-IL2, IL12-KS-IL2, and IL12-KS(Lightchain)+KS(Heavy chain)-IL2 proteins all showed potent IL-2 activity. Inaddition, in an ELISA, the IL12-KS-IL2 and IL12-KS(Light chain)+KS(Heavychain)-IL2 proteins both bound tightly to the EpCAM antigen, even thoughthe heavy and light chain V regions, respectively, are fused to otherproteins at their N-termini.

Example 7 Activity of Murine IL-12-GM-CSF Fusion Proteins

The IL-12 activity of a murine Fc-IL-12-GM-CSF molecule was tested in acell proliferation assay (FIG. 10). Human PBMCs were obtained from threevolunteers and cultured with 5 micrograms/ml phytohemagglutinin-P forthree days, washed with Hank's HBSS, and plated into microtiter platesat 10⁵ cells per well, according to a standard procedure (Gately, M. K.,Chizzonite, R., and Presky, D. H. Current Protocols in Immunology [1995]p. 6.16.1–6.16.15). Cells were incubated in the presence of various testproteins for 48 hours, and 0.3 microCuries of ³H-thymidine was added tenhours before determining levels of radioactive incorporation. IL-12 andan equimolar mixture of IL-12 and GM-CSF stimulated incorporation of³H-thymidine into cells in a dose-dependent manner, and the14.18-IL-12-GM-CSF fusion protein was about equally effective instimulating incorporation of ³H-thymidine. GM-CSF did not stimulateincorporation of ³H-thymidine at the concentrations tested, indicatingthat the observed incorporation of ³H-thymidine stimulated by the14.18-IL-12-GM-CSF fusion protein was due primarily to its IL-112activity.

In addition, the biological activity of the GM-CSF moiety of variousIL-12-Gm-CSF fusion proteins is tested in cell based assays. It is foundthat the GM-CSF moiety is active, with an activity per mole in the samegeneral range as commercial GM-CSF. For example, the biological activityof the GM-CSF moiety is tested in a different cell proliferation assay,following a procedure known to those practiced in the art of molecularimmunology (Cooper, S. C., and Broxmeyer, H. E. Current Protocols inMolecular Immunology [1996] p. 6.4.1–6.4.20). The mouse 32D(GM) cellline depends on GM-CSF for proliferation; this line has been adaptedfrom the original 32D cell line, described by Cooper and Broxmeyer, tobe particularly sensitive to GM-CSF (Faas et al., Eur. J. Immunol.[1993] 23:1201–14). The 32D(GM) cell line is not responsive to IL-12.32D(GM) cells in active log-phase growth are washed twice in mediumlacking GM-CSF and plated at about 5×10³ cells per well in microtiterwells in the presence of various amounts of commercial murine GM-CSF ormurine IL-12-GM-CSF fusion protein and grown for 48 hours. 0.3microCuries of ³H-thymidine are added sixteen hours before determininglevels of radioactive incorporation. There is a dose responsive increasein incorporation of ³H-thymidine with increasing levels of IL-12-GM-CSFfusion protein, indicating that the GM-CSF moiety of the IL-12-GM-CSFfusion protein is active. Moreover, the GM-CSF biological activity ofthe fusion protein, calculated on a molar basis, is comparable to thatof commercial murine GM-CSF.

Example 8 Treatment of Colon Carcinoma in an Immune-Proficient Mammalwith a Multiple Cytokine Fusion Protein

To test whether a multiple cytokine-antibody fusion protein could beused to treat colon carcinoma in a mammal with an intact immune system,the following experiments were performed. CT26 is a colon carcinoma cellline derived from Balb/C mice. By standard genetic engineeringtechniques, this cell line was engineered to express the humanepithelial cell adhesion molecule (EpCAM), which is the antigenrecognized by the KS-1/4 antibody; these cells are termed CT26/KSAcells.

Balb/C mice were subcutaneously inoculated with 2×10⁶ CT26/KSA cells.When tumors reached a volume of about 100–200 cubic millimeters, micewere randomized into three groups of 9 mice for further study. Beginningat day 0, tumor-bearing mice were treated with PBS, about 3.4 microgramsof KS-IL2 mixed with about 5.3 micrograms of KS-IL12, or about 6micrograms of KS-IL2-IL12. These doses are designed to deliver an equalnumber of IL-12 and IL-2 molecules to each set of mice. Mice wereinjected intratumorally, once per day for five days. Tumor sizes weremeasured with calipers.

The results of one such experiment are shown in FIG. 11. In thisexperiment, KS-IL12-IL2 caused a profound inhibition of tumor growth.The mixture of KS-IL12 and KS-IL2 also caused a significant inhibitionof tumor growth, but not as complete as KS-IL12-IL2. In the group ofmice treated with KS-IL12-IL2, six of nine mice were apparently cured oftheir tumors: these six mice survived until day 93, when the experimentwas terminated; and the tumors in these mice shrank and disappeared, sothat no subcutaneous tumor could be detected from day 39 to day 93. Theother three mice had tumors whose growth was delayed such that the tumorvolumes exceeded 4,000 cubic millimeters only after day 87.

Of the mice treated with a mixture of KS-IL12 and KS-IL2, two mice wereapparently cured of their subcutaneous tumors and survived until the endof the experiment. The tumors in the remaining seven mice did notdisappear and eventually grew to volumes of 1,000 cubic millimeter (1mouse) or greater than 4,000 cubic millimeters (6 mice).

The fact that KS-IL12-IL2 is more effective that an equimolar mixture ofKS-IL12 and KS-IL2 is surprising. The doses in this experiment deliverabout 15 picomoles of fission protein per dose, which corresponds toabout 9×10¹² molecules. At the start of treatment, each tumor has avolume of about 160 cubic millimeters, which corresponds to about 160million cells. Each cell expresses about 10⁶ molecules of EpCAM, sothere are about 1.6×10¹⁴ EpCAM antigen molecules to which the KSantibody might bind. Thus, when KS-IL12 and KS-IL2 were mixed andinjected into mice bearing such tumors, it is unlikely that these twoimmunocytokine fusion proteins competed with each other for antigenbinding sites. Thus, the effective dose of IL-12 and IL-2 at the tumorsite should have been at least as high for the mixture of KS-IL12 andKS-IL2 as for KS-IL12-IL2.

Example 9 Treatment of Colon Carcinoma in an Immunodeficient Mammal witha Multiple Cytokine Fusion Protein

Many forms of cancer therapy have the effect of killing dividing cells,including cells of the immune system. As a result, cancer patients oftenbecome immunosuppressed. To address whether multiple cytokine fusionproteins can be used to treat a mammal with a suppressed immune system,SCID mice bearing CT26/KSA tumors were treated with KS-IL12-IL2, amixture of KS-IL12 and KS-IL2, or PBS. SCID mice are deficient in both Bcells and T cells and depend on branches of the innate immune system,such as NK cells, for their ability to fight infections.

Mice with subcutaneous CT26/KSA tumors were generated as described inExample 8. Three groups of 8 mice each, bearing tumors of about 100 to200 cubic millimeters, were treated by intratumoral injection with thesame dosing and schedule as in Example 8. Results are shown in FIG. 12.In this case, the KS-IL12-IL2 fusion protein and the mixture of KS-IL12and KS-IL2 were about equally effective: five of eight mice were curedin each group by day 25. However, in the mice that were not cured, fiveof six tumors began to grow at a rate characteristic of tumors inuntreated animals, with an effective delay of about 14 to 21 days. Thisis in contrast to tumors in immune-proficient mice in Example 8: evenwhen tumors were not completely eliminated by treatment withKS-IL12-IL2, the tumors did not begin to grow aggressively until about60 days after the beginning of the experiment.

These experiments demonstrate that a multiple-cytokine antibody fusionprotein can be used to treat cancer in an immunosuppressed animal.

Example 10 Treatment of Lung Carcinoma by Intratumoral Injection of aMultiple Cytokine Fusion Protein: Comparison with Treatment byIndividual Immunocytokines

To address the effectiveness of multiple cytokine fusion proteins andimmunocytokines carrying single cytokine moieties against a lungcell-derived cancer, the following experiment was performed.

Lewis Lung Carcinoma (LLC) is an aggressive tumor derived from C57BL/6mice. An LLC cell line expressing the human EpCAM protein wasconstructed by standard genetic engineering techniques; the cell linewas termed LLC/KSA.

C57BL/6 mice with subcutaneous LLC/KSA tumors were generated asdescribed in Example 8 (check # of cells with KML). Four groups of 5mice each, bearing tumors of about 100 to 200 cubic millimeters, weretreated by intratumoral injection for five days. Mice were injected withPBS, about 20 micrograms of KS-IL 12, about 20 micrograms of KS-IL 12,or about 20 micrograms of KS-IL 12-IL2.

Results are shown in FIG. 13. In this case, the KS-IL12-IL2 fusionprotein was much more effective than either KS-IL12 or KS-IL2. In all ofthe mice treated with the KS-IL12-IL2 fusion protein, the tumorsdisappeared by day 27. On day 74, these mice were used in a lungmetastasis assay as described in Example 14; the original subcutaneoustumors did not reappear in the intervening period or during the secondexperiment. In contrast, treatment with either KS-IL2 or KS-IL12resulted in some apparent tumor shrinkage and a significant delay intumor growth, but the tumors did eventually grow. A comparison of theresults in this example and previous examples indicates that, forcertain diseases and modes of administration, treatment with a mixtureof immunocytokines carrying different cytokine moieties is superior totreatment with a single type of immunocytokine.

Example 12 Treatment of Lung Carcinoma by Intratumoral Injection of aMultiple Cytokine Fusion Protein: Comparison with Treatment by a Mixtureof Immunocytokines

To address the effectiveness of multiple cytokine fusion proteins andmixtures of immunocytokines carrying different cytokine moieties againsta lung cell-derived cancer, the following experiment was performed.

C57BL/6 mice with subcutaneous LLC/KSA tumors were generated asdescribed in Example 11. Three groups of 7 mice each, bearing tumors ofabout 100 to 200 cubic millimeters, were treated by intratumoralinjection for five days. Mice were injected with PBS, a mixture of about18 micrograms of KS-IL12 and about 11.5 micrograms of KS-IL12, or about20 micrograms of KS-IL12-IL2.

Results are shown in FIG. 14. In this case, the KS-IL12-IL2 fusionprotein was much more effective than the mixture of KS-IL12 and KS-IL2.In all of the mice treated with the KS-IL12-IL2 fusion protein, thetumors disappeared by day 27. In contrast, treatment with the mixture ofKS-IL12 and KS-IL2 resulted in some apparent tumor shrinkage and asignificant delay in tumor growth, but all of the tumors in thistreatment group did eventually regrow.

Example 13 Antigen-Dependence of Anti-Tumor Activity of a MultipleCytokine-Antibody Fusion Protein

To address the whether the effectiveness of a multiple cytokine-antibodyfusion protein in treatment of a tumor was dependent on thetumor-specific expression of the antigen recognized by the antibody, thefollowing experiment was performed.

A set of seven C57BL/6 mice with subcutaneous LLC/KSA tumors and asecond set of nine mice with tumors derived from the parental LLC cellline were generated as described in Example 11. These two groups ofmice, bearing tumors of about 100 to 200 cubic millimeters, were treatedby intratumoral injection for five days. Mice were injected with about20 micrograms of KS-IL12-IL2.

Results are shown in FIG. 15. In this case, the mice bearing the LLC/KSAtumors all were completely cured of their tumors. In contrast, only twoof the mice bearing the LLC tumors were cured; the other LLCtumor-bearing mice all enjoyed a transient reduction in their tumorvolumes, but their tumors eventually grew to large volumes.

These results indicate that the recognition of the EpCAM surface antigenpromotes the adherence of KS-IL12-IL2 to the surface of LLC/KSA tumorcells, and the resulting immune response is enhanced. Some anti-tumoreffect was observed against LLC-derived tumors as well; without wishingto be bound by theory, the antitumor effect of KS-IL12-IL2 in this casemay be due to the fact that the fusion protein was injected directlyinto the tumor and was therefore transiently localized to the tumor.

Example 14 Generation of an Immune Memory Against a Tumor Cell Type

The development of metastases is a major problem in treatment of cancer.To test whether treatment with a multiple cytokine antibody fusionprotein could lead to formation of a long-lasting immune memory againsta tumor cell type and could prevent the establishment of metastases, thefollowing experiment was performed.

Five C57BL/6 mice from Example 11 had been treated with KS-IL12-IL2, andhad apparently been cured of their subcutaneous tumors. On day 74relative to the initiation of treatment as described in Example 14,these five mice were injected i. v. with 10⁶ LLC/KSA cells. As acontrol, eight C57BL/6 mice were also injected i. v. with 10⁶ LLC/KSAcells.

On day 28, the mice were sacrificed and the lungs were examined formetastases. The lungs of the eight control mice were 70% to 100% coveredwith metastases, with an average of 85% lung surface coverage. The meanlung weight for these mice was 0.86 grams. In contrast, there were nometastases found on the surface of lungs from the five pre-treated mice,and the average lung weight was 0.28 grams, which corresponds to theweight of a normal mouse lung. These results indicated that thetreatment of the original tumor cells resulted in a long-lasting immunememory against the tumor cells; this memory prevented the establishmentof metastases of this tumor cell type.

TABLE X Protection of “regressed”mice from LLC-KSA pulmonary metastasesPrior Treatment Metastatic Score Lung Weight (g) None 4, 4, 4, 4, 4, 4,3, 3 0.88 +/− 0.27 KS-IL12/IL2 0, 0, 0, 0, 0 0.27 +/− 0.03The average lung weight of the control group, without tumor, was 0.2 g.Metastatic scores are based on % surface coverage of fused metastaticnodules where 0=no metastases; 1=1–25% coverage; 2=25–50% coverage;3=50–75% coverage; and 4=75–100% coverage

A second experiment to test for immune memory formation used six of theseven mice from Example 12 that had been injected with LLC/KSA tumorcells, had developed subcutaneous tumors, and had had those tumorsdisappear. Sixty-two days after the initiation of the treatment inExample 12, six pre-treated mice and 10 naive, untreated C57BL/6 controlmice were injected s. c. with 10₆ LLC cells. These cells do not expressthe human KS antigen, EpCAM.

In the naive mice, the injected LLC cells formed tumors that grew at arapid rate in all mice. In contrast, the tumors in the pre-treated micegrew much more slowly, and in one mouse, no subcutaneous tumor wasdetected. The results are shown in FIG. 16. Because the human KSantigen, EpCAM, is not expressed on LLC cells, the immune response tothe LLC cells was based on other antigens expressed by these cells.

Example 15 Multiple Cytokine Fusion Proteins as Vaccines

Multiple cytokine fusion proteins may be used as vaccines when fused toan antigen protein. The particular order of moieties from N-terminus toC-terminus, or whether the fusion protein is a single polypeptide chainor an oligomer, may vary depending on convenience of construction of theexpressing plasmids. The protein may be administered by a variety ofroutes, such as intravenous, subcutaneous, intraperitoneal, and so on.Similarly, the dose and frequency of administration generally need to beempirically determined, as is standard practice for human vaccines andas is well known to those skilled in the art of vaccine development.

For example, a fusion protein of the form antigen-IL-12-cytokine isadministered to a mouse, where the cytokine in the fusion protein is asecond cytokine different from IL-12. Control mice receive the sameamount of antigen-cytokine, antigen-IL-12, or antigen alone. At varioustimes during and/or after administration of the antigen fusion protein,blood samples are collected by retro-orbital bleeding and plasma isprepared and analyzed for the presence of antibodies directed againstthe antigen. It is found that antibodies are generated against theantigen. Moreover, the nature of the immune response to the antigen ischaracteristic of a Th1 response. The antibody response is stronger andthe type of antibodies produced are different than in certain controlimmunizations.

More specifically, a humanized antibody-murine IL-112-IL-2 fusionprotein in PBS buffer, is injected into Balb/c mice intravenously (5μg/day×5). Control mice receive the same antibody, in the same amounts,but with no attached IL12-IL-2. Neither injection solution contains anyother type of adjuvant. On day 10, blood samples are collected intomicrocentrifuge tubes by retro-orbital bleeding and plasma is preparedby collecting blood samples in plastic tubes containing sodium citrate,followed by centrifugation at full speed in an Eppendorf tabletopmicrocentrifuge. ELISA plates (96-well) are coated with the humanizedantibody protein, which contains the human constant region and is usedto capture any mouse antibodies made in response to the immunization.After washing away unbound material, the bound mouse antibodies aredetected with goat anti-mouse Fc antibody (Jackson ImmunoResearch)coupled to horse-radish peroxidase. Any bound antibodies could bedirected to either the human constant regions or the variable region,both of which are shared between the humanized antibody and the fusionproteins.

There is little or no reactivity to the humanized antibody without fusedIL-12-IL-2. The fusion protein, on the other hand, induces a strongantibody response in the absence of exogenous adjuvants and despite thefact that the intravenous route of administration is highly unfavorablefor inducing such responses, compared to either subcutaneous orintraperitoneal administration. Antibodies of the IgG2a isotype, whichare typical of IL-12-enhanced responses, are seen in theantibody-IL-12-IL-2 injected group but not the group injected with thehumanized antibody.

The immunogenicity of antigen-IL-12 multiple cytokine fusion proteinsadministered by various routes is tested by injecting a solution of thefusion protein (such as that described above) in PBS or otherbiocompatible buffer, or a known adjuvant such as Freund's incomplete orcomplete adjuvant. For example, single or multiple subcutaneous,intradermal or intraperitoneal injections can be given every two weeks.Alternatively, the fusion protein can be administered first bysubcutaneous injection and then followed by intraperitoneal injection.Freund's adjuvant cannot be used for human use, due to the irritation atthe injection site. Alternative adjuvants such as precipitates ofaluminum hydroxide (Alum) are approved for human use and can be used inthe present invention. New organic chemical adjuvants based on squalenesand lipids can also be used for injections into the skin.

Example 16 Gene Therapy with Multiple Cytokine Fusion Proteins

The anti-cancer activity of multiple cytokine fusion proteins deliveredby gene therapy methods was also demonstrated for treatment of lungcancer. Lewis Lung Carcinoma cells were stably transfected using theviral vector system described above (pLNCX-scIL-12-IL-2 or pLNCX-scIL-12DNA transfected into the PA317 packaging cell line). These constructsencode a single-chain version of IL-12, in which the p35 and p40subunits have been connected with a linker. Clones were selected invitro using G418-containing medium, and clones stably expressing about50 to 60 ng/ml of IL-12 were identified by ELISA (R & D Systems).

About 1×10⁶ and about 5×10⁶ LLC cells expressing scIL-12 or scIL-12-IL-2were injected s.c. into C57BL/6 mice and also into SCID mice. As acontrol, 2×10⁶ LLC cells were injected into C57BL/6 mice and also intoSCID mice. The LLC cells expressing IL-12 form tumors that grow at aboutthe same rate as tumors derived from LLC cells that have not beenengineered to express cytokines. However, in both C57BL/6 mice and alsoin SCID mice, LLC cells expressing scIL-12-IL-2 either did not formsubcutaneous tumors or formed tumors that subsequently shrank anddisappeared (FIGS. 17 and 18).

Example 17 Construction of Multiple Cytokine Fusion Proteins ContainingIL-4 and GM-CSF

The cytokines IL4 and GM-CSF, when used in combination, are potentactivators of dendritic cells. A multiple cytokine-antibody fusionprotein containing IL-4 and GM-CSF activity was constructed as follows.The coding sequence for GM-CSF was fused in-frame to the 3′ end of theKS-1/4 antibody heavy chain coding sequence, which was preceded by aleader sequence. In addition, the coding sequence for IL-4, including aleader sequence, was fused in-frame with a linker to the 5′ end of thecoding sequence for the mature KS-1/4 antibody light chain.

Specifically, to construct DNA encoding a fusion protein of murine IL-4and the light chain of the KS-1/4 antibody, the murine IL-4 cDNA wasadapted by PCR using the forward primer TCTAGACC ATG GGT CTC AAC CCC CAGC (SEQ ID NO:22), in which an XbaI site TCTAGA (residues 1–6 of SEQ IDNO:22) had been placed upstream of the translation initiation codon ATG,and the reverse primer C GGA TCC CGA GTA ATC CAT TTG CAT GAT GCT CTT TAGGCT TTC CAG G (SEQ ID NO:23), which contains a BamHI site GGA TCC(residues 2–7 of SEQ ID NO:23) immediately 3′ to the TCG codon(anticodon CGA) encoding the C-terminal amino acid residue of murineIL-4. After cloning of the PCR fragment and sequence verification, theXbaI-BamHI fragment containing the murine IL-4 cDNA was ligated to anBamHI-AflII oligonucleotide duplex encoding a flexible peptide linkerrich in glycine and serine residues. The AflII end was in turn joined toan artificially placed AflII site preceding the N-terminus of the matureKS-1/4 light chain. The DNA and protein sequences at the junctionsresulting from the two ligations are given below.

DNA:   TCG GGA TCC GGA GGT TCA GGG GGC GGA GGT AGC GGC GGA GGG GGC TCCTTA AGC GAG (SEQ ID NO:24) Protein: (Ser) Gly Ser Gly Gly Ser Gly GlyGly Gly Ser Gly Gly Gly Gly Ser Leu Ser (Glu) (SEQ ID NO:25)

In this DNA sequence, GGATCC (residues 4–9 of SEQ ID NO:24) and CTTAAG(residues 48–53 of SEQ ID NO:24) are the two restriction sites BamHI andAflII, respectively, used for reconstruction; TCG encodes the C-terminalserine residue of murine IL-4; GAG encodes the mature N-terminus of theKS-1/4 light chain; and the amino acid sequence of the GlySer-richpeptide linker is shown below the DNA sequence. Auxiliary sequences forhigh-level expression including strong promoters were placedappropriately around the DNA segments encoding both fusion polypeptides,using techniques described in previous examples and other standardtechniques of molecular biology.

The DNA sequences encoding the IL4-KS(light chain) and KS(heavychain)-GM-CSF fusion proteins were transfected into NS/0 cells and thecorresponding polypeptides were expressed at high levels. SDS-PAGE underreducing conditions showed a diffuse band at about 80 kD correspondingto the heavy chain-GM-CSF polypeptide and multiple bands at about 50 kDcorresponding to the IL4-light chain fusion. The appearance of diffuseand multiple bands were due to the variable glycosylation of IL-4 andGM-CSF, respectively.

The subunits assembled into a disulfide-bonded tetrameric protein with astructure corresponding to the generic structure in FIG. 3G. Thisprotein had the antigen-binding activity of KS-1/4, the ability to bindto Staph A protein via its Fc regions, and the cytokine activities ofIL-4 and GM-CSF. IL-4 activity was measured by IL-4-dependentstimulation of tritiated thymidine incorporation in CTLL-2 cells. GM-CSFactivity was measured by the GM-CSF-dependent stimulation of tritiatedthymidine incorporation in 32(D) GM cells. On a molar basis, the IL-4activity and the GM-CSF activity of KS-IL4-GMCSF were similar topurified IL-4 and GM-CSF.

A fusion of the cytokines IL-4 and GM-CSF, without associated antibodysequences, was constructed as follows. Murine IL-4 was cloned by PCRfrom RNA of murine spleen cells. The forward primer had the sequenceTCTAGACC ATG GGT CTC AAC CCC CAG C (SEQ ID NO:26), in which an XbaI siteTCTAGA (residues 1–6 of SEQ ID NO:26) was placed upstream of thetranslation initiation codon ATG, and the reverse primer had thesequence CGA TAT CCC GGA CGA GTA ATC CAT TTG CAT GAT GCT CTT TAG GCT TTCCAG G (SEQ ID NO:27), which placed an EcoRV site GAT ATC (residues 2–7of SEQ ID NO:27) immediately 3′ of the TCG codon (anticodon CGA)encoding the C-terminal amino acid residue of murine IL-4. Aftersequence verification, the XbaI-EcoRV fragment containing the muIL-4cDNA with its native leader was ligated to the SmaI-XhoI fragmentcontaining muGM-CSF cDNA to give the following sequence at the junctionof the fusion between muIL-4 and muGM-CSF: ATG, GAT TAC TCG TCC GGG ATGGGA AAA GCA CCC GCC CGC (SEQ ID NO:28), where the C-terminal sequence ofmuIL4 and the N-terminal sequence of muGM-CSF are in bold, and the G ATGGG (residues 17–22 of SEQ ID NO:28) is the sequence resulting from theligation of an EcoRV blunt end to a SmaI blunt end. The resultant DNAencoding muIL4-muGMCSF was then cloned into an expression vector. Theexpressed protein was analyzed by SDS-PAGE and found to run as a diffuseband with an apparent molecular weight of 45 to 50 kD. On a molar basis,the IL-4 activity and the GM-CSF activity of IL4-GMCSF were similar topurified IL-4 and GM-CSF.

Example 18 Construction of DNA Encoding Lymphotactin-KS-IL2 andExpression of the Lymphotactin-KS-IL2 Protein

Chemokines are a distinct class of cytokines that are thought to formgradients and mediate chemotaxis of immune cells. In addition, likeother cytokines, chemokines may induce expression of specific genes intarget cells. One characteristic of chemokines is that the freeN-terminus is often required for activity, which could place limits onthe ways that fusion proteins could be constructed.

A cytokine-antibody-cytokine fusion protein was constructed consistingof the cytokine lymphotactin, which is a chemokine, the antibody KS-1/4,and the cytokine IL-2. This fusion protein was tetrameric and comprisedtwo different polypeptides. One polypeptide consisted of murinelymphotactin fused to the N-terminus of the heavy chain of the KS-1/4antibody, followed by IL-2 at the C-terminus. The fusion of the KS-1/4heavy chain with IL-2 at the C-terminus, the “KS-IL2 heavy chain,” hasbeen described previously [Gillies et al. (1992) Proc. Natl. Acad. Sci.USA 89:1428]. The other polypeptide consisted of the light chain of theKS1/4 antibody.

The complete coding sequence of murine lymphotactin was published byKelner and Zlotnik (Science 266:1395 [1998]). To construct DNA encodinga fusion protein of murine lymphotactin and the heavy chain of theKS-IL2, the murine lymphotactin cDNA was adapted by PCR using theforward primer TCTAGAGCCACC ATG AGA CTT CTC CTC CTG AC (SEQ ID NO:29),in which an XbaI site TCTAGA (residues 1–6 of SEQ ID NO:29) was placedupstream of the translation initiation codon ATG, and the reverse primerGGA TCC CCC AGT CAG GGT TAC TGC TG (SEQ ID NO:30), which placed a BamHIsite GGA TCC (residues 1–6 of SEQ ID NO:30) immediately 3′ of the GGGcodon (anticodon CCC) encoding the C-terminal amino acid residue ofmurine lymphotactin. After cloning of the PCR fragment and sequenceverification, the XbaI-BamHI fragment containing the murine lymphotactincDNA was ligated to an BamHI-AflII oligonucleotide duplex encoding aflexible peptide linker rich in glycine and serine residues. The AflIIend was in turn joined to an artificial AflII site preceding the matureN-terminus of the KS-IL2 heavy chain. The DNA sequence at the junctionsresulting from the two ligations is given below:

    Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Ser(SEQ ID NO:32) CCC GGA TCC GGA GGT TCA GGG GGC GGA GGT AGC GGC GGA GGGGGC TCC TTA AGC CAG (SEQ ID NO:31)where GGATCC (residues 4–9 of SEQ ID NO:31) and CTTAAG (residues 48–53of SEQ ID NO: 31) are the two restriction sites BamHI and AflII,respectively, used for reconstruction; CCC encodes the C-terminal aminoacid residue of murine lymphotactin; CAG encodes the mature N-terminusof the KS-IL2 heavy chain; and the amino acid sequence of theGlySer-rich peptide linker is shown above the DNA sequence. The DNAencoding the murine lymphotactin-KS-IL2 heavy chain was then cloned intoan expression vector and then coexpressed with the KS 1/4 light chain.

The expressed lymphotactin-KS-IL2 fusion protein is tested forlymphotactin activity in a Boyden chamber migration assay using T cells(Leonard et al., [1999] Current Protocols in Immunology p. 6.12.3).Alternatively, NK cells are used. Alternatively, lymphotactin activityis observed in a standard cellular assay for calcium flux in response tothe activation of a G-protein coupled receptor (Maghazachi et al., FASEBJ. [1997];11:765–74.). In addition, the lymphotactin-KS-IL2 fusionprotein is tested and found to be active in assays for the ability tobind to EpCAM and is also active in assays for IL-2 activity, such asthe CTLL-2 cell proliferation assay.

Incorporation by Reference

All publications mentioned hereinabove are incorporated by referenceinto this application in their entirety.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

1. A fusion protein comprising: a first polypeptide chain comprising thep35 subunit of interleukin-12 (IL-12); and a second polypeptide chaincomprising a cytokine and the p40 subunit of IL-12, wherein the cytokineis a four-helix bundle protein.
 2. The fusion protein of claim 1,wherein the first polypeptide chain is covalently bonded to the secondpolypeptide chain.
 3. The fusion protein of claim 2, wherein the firstpolypeptide chain is covalently bonded to the second polypeptide chainby a disulfide bond.
 4. The fusion protein of claim 1, wherein thecytokine is interleukin-2 (IL-2).
 5. The fusion protein of claim 1,wherein the cytokine is granulocyte-macrophage colony stimulating factor(GM-CSF).
 6. The fusion protein of claim 1, wherein the cytokine isselected from the group consisting of interleukin-4 (IL-4),interleukin-6 (IL-6), and macrophage colony-stimulating factor (M-CSF).7. A nucleic acid encoding the fusion protein of claim
 1. 8. An isolatedor cultured cell comprising the nucleic acid of claim
 7. 9. A nucleicacid encoding the fusion protein of claim
 4. 10. An isolated or culturedcell comprising the nucleic acid of claim
 9. 11. A fusion proteincomprising a single-chain polypeptide comprising: the p35 subunit ofinterleukin-12 (IL-12); the p40 subunit of IL-12; and a second cytokine,wherein the second cytokine is a four-helix bundle protein, and whereinthe p35 subunit of IL-12, the p40 subunit of IL-12, and the secondcytokine are fused together as portions of a single, expressedpolypeptide chain.
 12. The fusion protein of claim 11, wherein thesecond cytokine is interleukin-2 (IL-2).
 13. The fusion protein of claim11, wherein the second cytokine is granulocyte-macrophage colonystimulating factor (GM-CSF).
 14. The fusion protein of claim 11, whereinthe second cytokine is selected from the group consisting ofinterleukin-4 (IL-4), interleukin-6 (IL-6), and macrophagecolony-stimulating factor (M CSF).
 15. A nucleic acid encoding thefusion protein of claim
 11. 16. An isolated or cultured cell comprisingthe nucleic acid of claim
 15. 17. The fusion protein of claim 11comprising, in an N- to C-terminal direction, the p35 subunit of IL-12,the p40 subunit of IL-12, and the second cytokine.
 18. A nucleic acidencoding the fusion protein of claim
 12. 19. An isolated or culturedcell comprising the nucleic acid of claim
 18. 20. A fusion proteincomprising: a first polypeptide chain comprising the p40 subunit ofinterleukin-12 (IL-12); and a second polypeptide chain comprising acytokine and the p35 subunit of IL-12, wherein the cytokine is afour-helix bundle protein.
 21. The fusion protein of claim 20, whereinthe first polypeptide chain is covalently bonded to the secondpolypeptide chain.
 22. The fusion protein of claim 21, wherein the firstpolypeptide chain is covalently bonded to the second polypeptide chainby a disulfide bond.
 23. The fusion protein of claim 20, wherein thecytokine is interleukin-2 (IL-2).
 24. The fusion protein of claim 20,wherein the cytokine is granulocyte-macrophage colony stimulating factor(GM-CSF).
 25. The fusion protein of claim 20, wherein the cytokine isselected from the group consisting of interleukin-4 (IL-4),interleukin-6 (IL-6), and macrophage colony-stimulating factor (M-CSF).26. A nucleic acid encoding the fusion protein of claim
 20. 27. Anisolated or cultured cell comprising the nucleic acid of claim
 26. 28. Anucleic acid encoding the fusion protein of claim
 23. 29. An isolated orcultured cell comprising the nucleic acid of claim 28.